Preparation, Structural Characterization And Functional Evaluation Of Milk Protein-EGCG And Chitosan-chlorogenic Acid Complexes

The objectives of the present study were to first covalently modify different bovine milk proteins (a-lactalbumin,3-lactoglobulin, lactoferrin and sodium caseinate) using (-)-epigallocatechin-3-gallate (EGCG), then compare structural and functional properties between covalent and non-covalent protein-EGCG complexes and finally test the complexes with greater antioxidant potential in stabilizing a model P-carotene emulsion. Another objective of the present study was to investigate influence of covalent chitosan-(-)-epigallocatechin-3-gallate (EGCG) complexes on physicochemical properties of β-carotene emulsions coated by a-lactalbumin (LA) or sodium caseinate (SC). Afterward, covalent modification of chitosan with EGCG was achieved by hydroxyl-free radical grafting. Then based on layer-by-layer electrostatic deposition, bilayer emulsions stabilized with different milk proteins and chitosan-EGCG conjugates (CEC) were prepared. The physicochemical properties of primary and secondary emulsions were characterized and compared. Finally, chitosan-chlorogenic acid (CA) covalent complex was synthesized, and structural and functional properties of chitosan-CA covalent complex and physical complex were compared. Then ability of chitosan-CA complexes in stabilizing a model β-carotene emulsion was evaluated. The main results were listed as follows:1. Covalent modification of milk proteins with EGCG was testified by a reduction of free amino groups and free sulfhydryl groups as well as matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proposed schematic formation pathway showed that milk proteins formed covalent complexes with EGCG dimers. In terms of functional properties, covalent protein-EGCG complexes had higher denaturation temperatures than non-covalent ones. Besides, covalent protein-EGCG complexes exhibited much stronger antioxidant activity than the same amount of non-covalent ones.2. Covalent protein-EGCG complexes exhibited better interfacial adsorption behavior and greater antioxidant potential than non-covalent ones in emulsion systems. Covalent protein-EGCG complexes significantly enhanced chemical stability of P-carotene in emulsions against heat treatment and ultraviolet (UV) light exposure. Given both physical and chemical stability of emulsions, the overall ability of covalent protein-EGCG complexes in stabilizing a model β-carotene emulsion followed the order:covalent SC-EGCG complex> covalent LG-EGCG complex> covalent LF-EGCG complex> covalent LA-EGCG complex.3. Physical stability of SC-coated emulsion in the absence or presence of environmental stresses was improved by the adsorption of CEC, while the layer-by-layer electrostatic deposition did not improve the stability of emulsions stabilized by LA. Besides, steady-state flow measurements revealed that the viscosity of primary emulsions could be enhanced by the adsorption of CEC. In comparison with the primary emulsions, the bilayer emulsions significantly (p<0.05) improved chemical stability of β-carotene against heat treatment and ultraviolet (UV) light exposure, and the least degradation of3-carotene occurred in SC-CEC coated emulsion. In general, SC-CEC coated emulsion exhibited the best physicochemical properties among all emulsions studied in this research.4. Expermiments of FTIR, UV-vis and NMR confirmed that chitosan-CA covalent complex was synthesized. The covalent reaction induced changes of chemical constitution in chitosan, and conjugation of CA onto chitosan apparently resulted in formation of both amide and ester bonds. XRD and SEM results indicated that conjugation of CA changed crystallinity and morphology of chitosan, and chitosan-CA covalent complex induced larger changes of physical structure than chitosan-CA physical complex. In terms of functional properties, chitosan-CA covalent complex exhibited higher antioxidant activity than chitosan-CA physical complex. The viscosity of chitosan solution was significantly increased after conjugation of CA.5. In comparison with emulsions stabilized by chitosan and chitosan-CA physical complex, emulsions coated by chitosan-CA covalent complex had smaller droplet size and higher viscosity. The physicochemical stability of emulsion stabilized by chitosan-CA covalent complex was better than those stabilized by chitosan and chitosan-CA physical complex.