Study Of The Interaction Mechanisms And Functional Properties Of Lactoferrin-Plant Active Molecules Non-covalent Complexes

China has gone into the Age of Big Health with the in-depth development of nutrition science,and the functions and applications of milk ingredients have attracted increasing attentions.However,the categories of dairy products in China are fewer and more homogenized,optimizing the structure of dairy products and developing more functional dairy products are advocated in the future development of China's dairy industry.New milk protein-based products and precision nutrition will be the trend and research focus in the dairy research.Lactoferrin,as an active protein in milk,has a variety of surface properties and multifunctional activities,thus being well recognized by consumers and exhibiting great potentials in scientific and industrial area.Plant active molecules play an important role in disease prevention and human health,but their poor water solubility severely decreased their bioavailability and limited their development in the related industries.The in vitro stability and in vivo targeted-delivery of plant active molecules are crucial scientific issues to be addressed.In order to establish dairy-derived functional carrier systems and develop functional dairy products,in this thesis we used lactoferrin to prepare lactoferrin-plant active molecule non-covalent complexes,revealed the non-covalent interaction mechanism between lactoferrin and plant active molecules,further explored the physicochemical properties and functional activities of lactoferrin-plant active molecule complexes,and finally developed lactoferrin-ginsenoside Rg₃ oil-in-water nano-emulsions,evaluating the potential of lactoferrin as a delivery carrier.This thesis provides a theoretical basis and new example for understanding the binding and interaction mechanisms of lactoferrin and plant active molecules,as well as expanding the development of functional dairy products.The main research contents and conclusions of this article are listed below:(1)We prepared lactoferrin-ginsenoside Rg₃ non-covalent complex(LF-Rg₃),and further prepared LF-Rg₃ oil-in-water nano-emulsions,of lactoferrin and ginsenoside Rg₃,suggesting the potential of lactoferrin as a delivery carrier.We investigated the interaction mechanism of lactoferrin and ginsenoside Rg₃ as well as the changes of lactoferrin secondary structure by UV spectroscopy,fluorescence spectroscopy,circular dichroism,and surface hydrophobicity.It was shown that ginsenoside Rg₃ could quench the endogenous fluorescence of lactoferrin and spontaneously formed a complex with a molar ratio of 1:1.The interaction between lactoferrin and ginsenoside Rg₃ were studied by thermodynamic analysis and molecular docking,indicating that hydrogen bonding and van der Waals forces are the main driving forces of their interactions.Finally,the microstructure,storage stability,and cytotoxicity of LF-Rg3 oil-in-water nano-emulsions were evaluated.It was found that the introduction of ginsenoside Rg₃did not significantly change its properties except for the formation of a very small amount of large droplet particles during storage.(2)We prepared lactoferrin-ginsenoside Re/Rb₁ non-covalent interaction,we found that ginsenoside Re/Rb₁ can enhance the foaming properties of lactoferrin.We investigated the interaction pattern of lactoferrin and ginsenoside Re/Rb₁ and the changes of lactoferrin secondary structure by UV spectroscopy,fluorescence spectroscopy,circular dichroism,and surface hydrophobicity.It was shown that ginsenoside Re/Rb₁ could quench the endogenous fluorescence of lactoferrin and spontaneously formed a complex with a molar ratio of 1:1.The interaction mechanism between lactoferrin and ginsenoside Re/Rb₁ were studied by thermodynamic analysis and molecular docking,indicating that hydrogen bonding and van der Waals forces are the main driving forces of the interactions.Finally,we investigated the effect of different chemical structures of ginsenosides on the physicochemical properties of proteins,and tested the emulsification and foaming properties of the complexes.It was found that the addition of ginsenosides did not significantly affect the emulsification activity of lactoferrin,but the emulsification stability of lactoferrin decreased from219.17 min and 165.35 min to 89.17 min and 90.20 min after forming noncovalent complex with Re/Rb₁ respectively.The foaming properties of lactoferrin increased from141%and 150%to 200.67%and 189.5%,respectively,while there was a slight downward trend in foam stability.(3)We prepared lactoferrin-thymol non-covalent complex,and found the increased antibacterial ability against E.coli and S.aureus compared to lactoferrin alone.We investigated the interaction mechanisms,changes in the secondary structure of lactoferrin,and characterized the generation of lactoferrin-thymol complexes by UV spectroscopy,fluorescence spectroscopy,circular dichroism,and scanning electron microscopy.We investigated the interaction forces between lactoferrin and thymol through molecular docking and kinetic simulations and kinetic simulations indicating three possible binding sites and five predicted stable binding modes for lactoferrin to thymol,facilitated by hydrogen bonding and hydrophobic interactions.Finally,the antimicrobial effect of lactoferrin-thymol complex was investigated by measuring the minimum inhibitory concentration,growth curve and bacterial inhibitory activity.It was shown that LF-Thy complex exhibited significant antibacterial ability against E.coli and S.aureus compared to lactoferrin.The minimum inhibitory concentrations of lactoferrin against E.coli and S.aureus were>40 mg/m L and 40 mg/m L,which decreased to 10 mg/m L and 5 mg/m L after combined with thymol,respectively.This work contributed to further understanding of LF-Thy complex interactions and demonstrated the potential antibacterial activity of the LF-Thy complex.(4)We prepared lactoferrin-resveratrol/pterostilbene non-covalent complexes,and found the increased antioxidant properties of lactoferrin complexes compared lactoferrin alone.We investigated the interaction mechanisms of lactoferrin with resveratrol/pterostilbene,changes in the secondary structure of lactoferrin,and characterization of the generation of lactoferrin-resveratrol/pterostilbene complexes by UV spectroscopy,fluorescence spectroscopy,circular dichroism,quenching constants,and scanning electron microscopy.The experiments showed that resveratrol/pterostilbene could quench the endogenous fluorescence of lactoferrin,and spontaneously form complexes with lactoferrin with a molar ratio of 1:1.The interaction forces between lactoferrin and phenolic compounds of different structures were investigated by thermodynamic analysis and molecular docking methods.Experiments showed spontaneous binding under hydrogen bonding and van der Waals forces.From the combined free energy calculations,the main driving force in the LF-Res system is the hydrogen bonding interaction,while the main driving force in the LF-Pte system is the van der Waals interaction.Resveratrol showed stronger binding capacity and more binding sites to lactoferrin compared to pterostilbene.Finally,the antioxidant properties of lactoferrin complexes were investigated,showing that the antioxidant properties of lactoferrin complexes with resveratrol/pterostilbene were enhanced,which might be due to the fact that lactoferrin could enhance the solubility of resveratrol/pterostilbene in solution.