Construction And Application Of The Antioxidant Carrier: Egg White Protein-polyphenol Conjugates

Most of functional factors are poor in water-solubility,sensitive to light or heat,prone to oxidize and degrade and have low bioaccessibility.Therefore,it is of great significance to construct an effective food functional factor delivery system for the homeostasis of functional factors and the improvement of their bioavailability.In this paper,ultrasound-assisted free radical method was used to prepare egg white protein（EWP）–polyphenol（Caffeic acid,CF,Chlorogenic acid,CA,Epigallocatechin-3-gallate,EGCG,Catechin,CT,Quercetin,QE）conjugates.The regulation and control mechanism of ultrasound-assisted on reaction process and the grafting sites of EWP with different polyphenols were studied,in order to reveal the effects of ultrasound or characteristics of polyphenols on the structural and functional properties of EWP.And then antioxidant carrier（EWP–CF）was screened and applied to the preparation,stability and simulation digestion in vitro of fish oil emulsion.The contents and conclusions are listed as follows:（1）The effect of ultrasound and polyphenols covalent modification on the structural and functional properties of EWP–polyphenol conjugates was studied to reveal the correlation between the functional properties and structure of EWP–polyphenol conjugates.The results showed that the grafting of EWP to polyphenols was greatly accelerated by ultrasound treatment（the time reduced from 24 h to 1 h）.Compared with the molecular weight of selected polyphenols,the structure of polyphenols（position and number of phenolic hydroxyl groups）had more important effects on the grafting degree of polyphenols to EWP.In addition,polyphenol grafting significantly decreased the contents ofα-helix,and increased the contents ofβ-sheet and surface hydrophobicity（H₀）（P<0.05）,on the other hand,it also significantly improved the antioxidant,emulsifying activity and thermal stability of EWP（P<0.05）.The results of correlation analysis showed that ABTS·+scavenging rate and ferric reducing activity were negatively correlated with content ofα-helix,and positively correlated with content ofβ-sheet,T-turn and random coil（P<0.05）,EAI and Tmax were positively correlated to H₀（P<0.05）.In conclusion,ultrasound-assisted free radical grafting is a green and efficient technology of EWP to polyphenols,which could be applied to the preparation of EWP–polyphenols antioxidant emulsifier.（2）Take EWP–polyphenol conjugates as a model,traditional chemical analysis methods and high performance liquid chromatography-mass spectrometry（HPLC–MS）were combined to determine the grafting sites of EWP–polyphenol conjugates at the molecular level,revealing the covalent reaction mechanism between EWP and polyphenols under traditional water bath and ultrasound treatment.The results showed that phenolic hydroxyl groups of polyphenols were covalent grafted to EWP by C-N and C-S bonds,which reduced the contents of free amino,thiol and free tryptophan groups in EWP.Polyphenols with a structure C₆-C₃ had higher reactivity with His in EWP,while polyphenols with a structure C₆-C₃-C₆ had higher reactivity with Asp,Ser,Glu and His in EWP,and the number of amino acids involved in the reaction increased under ultrasound treatment,which were usually hydrophobic amino acids.The number of grafting sites was significantly increased by 17–57 under ultrasound treatment compared with water bath.In addition,albumin,which accounted for the largest number of EWP,had a higher reaction degree of EWP with polyphenols,and ultrasound treatment increased the number of graft sites of albumin and types of polyphenols involved in the reaction.In conclusion,the types of polyphenol had different affinity with the components of EWP,and more grafting sites were involved under ultrasound treatment compared to water bath,therefore speeding up the covalent reaction.（3）The fish oil emulsions were prepared by using EWP and EWP–CF conjugate（excellent antioxidant activitie and emulsifying propertie）as emulsifiers,and then the structure,theological properties,physicochemical stability and digestive properties of fish oil emulsions were investigated.The results showed that the fish oil emulsion prepared by EWP–CF conjugate not only had a smaller particle size and was evenly dispersed,but also better physicochemical stability due to its outstanding antioxidant.And the improvement of oxidation stability is the most significant,TBARS only changed from 3.42 to 5.21 mg/kg after 15 days,while that of EWP emulsion increased from 4.64 mg/kg to 20.35 mg/kg.In addition,the fish oil emulsion prepared by EWP–CF conjugate had a slower digestion rate than control,and the lower digestion rate in the stomach could protect the fish oil from destruction.