| Whey protein(WP)is a byproduct of cheese production,with high nutritional value and functional properties,widely used in the food industry.Chlorogenic acid(CA)is an important bioactive substance with antibacterial,antiviral,anti-tumor,and antioxidant effects.Pectin mainly exists in the form of high methoxy pectin(HMP)in nature.HMP has good hydration and flexibility,and the presence of acetyl groups enhances its interfacial activity,making it suitable for stable delivery systems.Whey protein,chlorogenic acid,and high methoxy pectin are not only renewable and safe but also biocompatible.Currently,research on the interaction mechanisms and structural characterization of whey protein,chlorogenic acid,and high methoxy pectin in nanocomposites constructed based on non-covalent and covalent interactions is limited.Moreover,there is little research on the influence of different addition and grafting sequences of non-covalent and covalent complexes on the stability and functionality of delivery systems.This study,supported by the National Natural Science Foundation of China Youth Science Foundation Program(Project No.32101881),employed noncovalent and covalent reaction methods to synthesize binary and ternary complexes of whey protein,chlorogenic acid,and high methoxy pectin.Various addition and grafting sequences were explored to elucidate the reaction mechanisms and assess the functional properties of the complexes.Moreover,these complexes were employed as the foundational materials for crafting an active factor delivery system,with a focused examination into their stabilizing effects on the functional components of the delivery system.The results of the research not only provide technical assurance but also offer theoretical underpinnings for the precise design and establishment of reliable and robust functional delivery systems.Additionally,the findings offer novel insights for enhancing the application value of whey protein,polyphenols,and pectin in the food industry.The main research content and results of this article are as follows:(1)Spectroscopic analysis and molecular docking techniques were employed to study the non-covalent binding interactions between three types of whey proteins,namely β-lactoglobulin(β-Lg),α-lactalbumin(α-La),bovine serum albumin(BSA)and chlorogenic acid.The spectroscopic study results indicated that chlorogenic acid quenched the fluorescence of the three whey proteins via a static quenching mechanism.The number of binding sites for chlorogenic acid on the three proteins was approximately equal to 1,and the binding affinities followed the order of α-La > β-Lg >BSA.Thermodynamic parameters revealed the hydrophobic forces in the three systems.Fluorescence resonance energy transfer measurements showed that the probability of energy transfer between the three whey proteins and chlorogenic acid was α-La > BSA >β-Lg.Surface hydrophobicity followed the order of α-La > β-Lg > BSA.The structures of α-La and β-Lg became more flexible after interacting with chlorogenic acid,whereas the structure of BSA became denser.Molecular docking models suggested that the most likely binding sites for β-Lg,α-La and BSA were the outer surface,crevice and substructure domain I,respectively.Hydrophobic interactions and hydrogen bonding between α-La,β-Lg and chlorogenic acid contributed to the formation of non-covalent complexes between chlorogenic acid and whey proteins.(2)Based on the results of electrostatic complexation between whey protein and high methoxy pectin,non-covalent ternary complexes were prepared using whey protein,high methoxy pectin and chlorogenic acid at p H 4,4.5,and 5.The structure and functional properties of these nanocomplexes were characterized.Fluorescence quenching results showed that the binding constants between whey protein and high methoxy pectin complexes to chlorogenic acid increased with increasing p H values.The ternary complexes exhibited good antibacterial and antioxidant activities,with antibacterial activity increasing as the p H decreased and antioxidant activity increasing as the p H increased.Based on these findings,whey protein,chlorogenic acid and high methoxy pectin binary and ternary complexes were prepared at p H 4.5 under different addition sequences.Spectroscopic and molecular docking techniques were used to study the intermolecular interactions in the system.It was found that hydrogen bonding,electrostatic interactions and hydrophobic forces were present between molecules in the ternary system.High methoxy pectin exhibited more significant hydrophobic interactions with whey protein than chlorogenic acid,which could have an important impact on the conformation and stability of the samples.Subsequently,lycopene-loaded emulsions were prepared using the complexes and characterized.The results showed that the ternary complex emulsions outperformed binary complex emulsions in terms of photostability,thermal stability,storage stability,lycopene retention,and antioxidant tests.Among the different addition sequences,the HMP-CA-WPI complex exhibited the best performance.This indicates that the order of addition of whey protein,chlorogenic acid,and high methoxy pectin not only affects the interactions and structural properties of the complexes but also allows for the modulation of the functional properties of the emulsions.(3)Covalent ternary complexes of whey protein,chlorogenic acid and high methoxy pectin were prepared using the Maillard reaction and alkaline grafting methods with different grafting sequences.The study aimed to investigate the reaction mechanism of covalent complexes and evaluate their functional properties.SDS-PAGE Gel electrophoresis results confirmed the formation of covalent complexes.The covalent sequence WPI-CA-HMP showed a high degree of pectin grafting,while the WPI-HMP-CA sequence had a high chlorogenic acid binding equivalent.This indicates that glycosylation and alkaline grafting promote the covalent grafting of whey protein with chlorogenic acid and high methoxy pectin.SEM results demonstrated that the microstructure of whey protein underwent varying degrees of changes after covalent grafting with high methoxy pectin.Scanning images of the WPI-CA complex indicated that chlorogenic acid promoted the aggregation and cross-linking of whey protein.Spectral experiments revealed changes in the secondary and tertiary structures of whey protein after covalent modification.The free radical scavenging ability significantly increased in both binary and ternary covalent complexes,with the ternary complexes showing stronger free radical scavenging ability.These research findings can expand new application directions for the covalent binding of proteins,polyphenols and polysaccharides in the development of functional foods.(4)The construction of covalent binary and ternary complexes involving whey protein,chlorogenic acid and high methoxy pectin for the encapsulation of lycopene in emulsions was carried out.The study investigated the impact of covalent complexes on the stability of the emulsion and their behavior during in vitro digestion.The results indicated that both binary and ternary covalent complexes enhanced the physicochemical stability of the emulsion against p H,ultraviolet light,salt ions,and heat treatment.The protective effects of the two types of ternary covalent complexes were superior,regardless of the covalent sequence.In terms of emulsion storage stability,there were no significant changes in the average particle size and zeta potential.The retention rate and color difference rate of lycopene in the ternary covalent emulsion were significantly better than those in the binary emulsion.The emulsion stabilized by ternary covalent complexes exhibited lower release rates of fatty acids and lower bioavailability of lycopene compared to the binary system,indicating a notable delayed release effect in the intestinal tract.This study offers valuable theoretical insights for the purposeful design and establishment of secure and reliable functional delivery systems. |
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