lactoferrin and epigallocatechin-gallate

The aim of this study was to prepare lactoferrin-epigallocatechin-3-gallate (LF-EGCG) conjugates and to determine their ability to protect emulsified algal oil against aggregation and oxidation. LF-EGCG conjugates were formed using an ultrasound-assisted alkaline treatment. The ultrasonic treatment significantly improved the grafting efficiency of LF and EGCG and shortened the reaction time from 24 h to 40 min. Fourier transform infrared spectroscopy and circular dichroism spectroscopy analyses showed that the covalent/non-covalent complexes could be formed between LF and EGCG, with the CO and CN groups playing an important role. The formation of the conjugates reduced the α-helix content and increased the random coil content of the LF. Moreover, the antioxidant activity of LF was significantly enhanced after conjugation with EGCG. LF-EGCG conjugates as emulsifiers were better at inhibiting oil droplet aggregation and oxidation than LF alone. This study demonstrates that ultrasound-assisted formation of protein-polyphenol conjugates can enhance the functional properties of the proteins, thereby extending their application as functional ingredients in nutritionally fortified foods.

Topics: Antioxidants; Catechin; Emulsions; Lactoferrin; Polyphenols

The impact of covalent attachment of (-)-epigallocatechin gallate (EGCG) to lactoferrin (LF) on the structure, morphology, functionality, and allergenicity of the protein was studied. These polyphenol-protein conjugates were formed using various enzymatic (laccase- and tyrosinase-catalyzed oxidation) and nonenzymatic (free radical grafting and alkali treatment) methods. The preparation conditions for forming the enzymatic conjugates were optimized by exploring the influence of order-of-addition effects: protein, polyphenols, and enzymes. The total phenol content of the LF-EGCG conjugates was quantified using the Folin-Ciocalteu method. The nature of the conjugates formed was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FTIR) spectroscopy analyses. These studies showed that enzymatic cross-linking was a highly effective means of forming LF-EGCG conjugates. Analysis of these conjugates using various spectroscopic methods showed that conjugation to EGCG changed the molecular structure of LF. Atomic force microscopy showed that the four covalent cross-linking methods affected the size and morphology of these LF-EGCG conjugates formed. The antioxidant activity and emulsifying stability of LF were significantly improved by conjugation to EGCG. Finally, an enzyme-linked immunosorbent assay (ELISA) and a western blot assay indicated that conjugation of EGCG reduced the binding capacity of LF to immunoglobulin E (IgE) and immunoglobulin G (IgG), which is consistent with a decrease in allergenicity. Overall, this study suggests that LF-EGCG conjugates formed using enzymatic or nonenzymatic methods have potential applications as functional ingredients in foods.

Topics: Allergens; Catechin; Lactoferrin; Polyphenols

Proteins, polysaccharides, and polyphenols can be assembled into ternary complexes with functional attributes that combine the desirable facets of each individual component. In the present work, we characterized the interactions among lactoferrin (LF), hyaluronic acid (HA) and (-)-epigallocatechin gallate (EGCG) dispersed in aqueous solutions at various pH values (2 to 9), HA levels (0 to 0.5 wt%), and EGCG levels (0 to 0.105 wt%). The antioxidant activity of the complexes was also evaluated. The ternary complexes formed macroscopic hydrogels at pH 2, microgel dispersions at pH 3, and soluble complexes at pH > 4. Information about the role of hydrogen bonds, electrostatic forces, and hydrophobic interactions was obtained using spectroscopic methods. The antioxidant activity of the ternary complexes was much higher at pH 5 than at pH 3, which was attributed to the ability of the hydrogels to inhibit the access of the free radicals generated in the aqueous phase to EGCG trapped inside. These results may be useful for optimizing the formulation of effective delivery systems for natural antioxidants.

Topics: Catechin; Cosmetics; Functional Food; Hyaluronic Acid; Lactoferrin; Technology, Pharmaceutical

The impact of lactoferrin (LF)-chlorogenic acid (CA) and (-)-Epigallocatechin-3-gallate (EGCG) conjugates on the physicochemical properties of β-carotene emulsions was investigated. Formation of lactoferrin-polyphenol conjugates, which was confirmed by SDS-PAGE, caused changes in the structure and nature of lactoferrin. Based on layer-by-layer electrostatic deposition, β-carotene bilayer emulsions were prepared by lactoferrin and lactoferrin-polyphenol conjugates at pH 7.0. The physicochemical properties of primary and secondary emulsions were evaluated and the results suggested that LF-polyphenol conjugates-stabilized primary and secondary emulsions exhibited better emulsifying properties and improved physical stability of β-carotene bilayer emulsions under freeze-thaw, ionic strength and thermal treatments. In addition, the lactoferrin-polyphenol conjugates could effectively enhance chemical stability of β-carotene in oil-in-water emulsions against heat treatment and ultraviolet light exposure, and the least degradation of β-carotene occurred in LF-EGCG conjugate-stabilized primary emulsion. The interfacial engineering technology utilized in this study may lead to the formation of emulsions with improved physicochemical and functional performance.

Topics: beta Carotene; Catechin; Chemical Phenomena; Chlorogenic Acid; Electrophoresis, Polyacrylamide Gel; Emulsions; Food Technology; Hydrogen-Ion Concentration; Lactoferrin; Polyphenols

The aggregation of lactoferrin and (-)-epigallocatechin gallate (EGCG) was inhibited by polyphenols, oligosaccharides, and collagen peptide in this study. Polyphenols, oligosaccharides, or collagen peptide can effectively prevent the formation of lactoferrin-EGCG aggregates, respectively. The addition sequence of lactoferrin, polyphenols (oligosaccharides or collagen peptide) and EGCG can affect the turbidity and particle size of the ternary complexes in the buffer solution; however, it hardly affected 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 surrounded the lactoferrin molecule surface with weaker binding affinities, forming polyphenols or a collagen peptide-lactoferrin-EGCG ternary complex; for neutral oligosaccharides, the ternary complex was generated mainly through steric effects, accompanied by a change in the lactoferrin secondary structure induced by gallic acid, chlorogenic acid, and xylo-oligosaccharide. Polyphenols, oligosaccharides, or collagen peptide restraining the formation of lactoferrin-EGCG aggregate could be applied in the design of clear products in the food, pharmaceutical, and cosmetic industries.

Topics: Catechin; Collagen; Food Additives; Humans; Lactoferrin; Oligosaccharides; Polyphenols; Protein Aggregates; Protein Structure, Secondary

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 pH 5.0. 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 decreased with an increase in 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. These findings should provide an insight into the fabrication mechanism and applications of ternary aggregates formed by protein, polysaccharide, and polyphenol in the food, pharmaceutical, and cosmetic industries.

Topics: Animals; Catechin; Cattle; Circular Dichroism; Fluorescence; Lactoferrin; Particle Size; Pectins; Protein Structure, Secondary; Viscosity

The interactions between native, thermally modified lactoferrin (LF) and (-)-epigallocatechin-3-gallate (EGCG) at pH 3.5, 5.0, and 6.5 were investigated. Turbidity, particle size, and charge of LF-EGCG complexes were mainly dominated by pH value and secondary structure of protein. At pH 3.5 and 5.0, LF-EGCG complexes were nanoparticles which had high ζ-potential, small size, and soluble state. At pH 6.5, they were submicrometer particles which exhibited low ζ-potential, large size, and insoluble state. The infrared spectra of freeze-dried LF-EGCG complexes showed that they were different from LF and EGCG alone. Far-UV CD results indicated that heat denaturation might irreversibly alter the secondary structure of LF and EGCG induced a progressive increase in the proportion of α-helix structure at the cost of β-sheet and unordered coil structure of LF at pH 3.5, 5.0, and 6.5. EGCG exhibited a strong affinity for native LF but a weak affinity for thermally modified LF at pH 5.0 and 6.5. An inverse result was observed at pH 3.5. These results could have potential for the development of food formulations based on LF as a carrier of bioactive compounds.

Topics: Catechin; Drug Carriers; Drug Stability; Hot Temperature; Hydrogen-Ion Concentration; Lactoferrin; Nanoparticles; Particle Size; Protein Binding; Protein Structure, Secondary

We found a novel procaspase-3 activating cascade mediated by lysosomal enzyme. The activating enzyme of procaspase-3, named lysoapoptase having the molecular weight of 78kDa was determined to be a lactoferrin located in the lysosome. Recombinant lactoferrin accelerated the processing of procaspase-3 to form active caspase-3 in vitro. D-Galactosamine is a well-known inducer of hepatocyte apoptosis. The caspase-3 which plays a common central role in the final step of various apoptosis cascades, was dramatically increased in the cytoplasm by the d-galactosamine administration in vivo. When D-galactosamine was administrated as a death signal in vivo, the lysosomal lactoferrin was released into the cytoplasm and procaspase-3 located in the cytoplasm was processed to form active caspase-3. The cotreatment of epigallo-catechin gallate resulted in the complete protection of the hepatocyte apoptosis suppressing the increases of caspase-3 in the cytoplasm. The caspase-3 activity was also inhibited directly by the epigallo-catechin gallate. Therefore, all apoptosis cascades mediated by caspase-3 should be suppressed by epigallo-catechin gallate. The caspase-3 activity was not only directly inhibited by epigallo-catechin gallate in vitro, but the release of lactoferrin from the lysosomes into the cytoplasm was also suppressed by epigallo-catechin gallate treatment in vivo. Therefore, the apoptosis induction was suppressed at the two successive steps by cotreatment of epigallo-catechin gallate in vivo.

Topics: Amino Acid Substitution; Animals; Apoptosis; Caspase 3; Caspases; Catechin; Cell Death; Galactosamine; Kinetics; Lactoferrin; Liver; Lysosomes; Models, Biological; Protease Inhibitors; Protein Processing, Post-Translational; Recombinant Proteins