Establishment Of A Model For The Interaction Of Phlorizin With Pepsin And Trypsin And Its Nanoparticles Preparation And Application In Yogurt

Phlorizin（PHL）is a flavonoid which mainly exists in apples and Lithocarpus polystachyus Rehd.It has a variety of health promotion effects,including antioxidant activity,cardiovascular protection and antiviral activity.When ingested by organisms,PHL may interact with digestive enzymes,which may affect the activity of digestive enzymes and the digestion and absorption of PHL.At present,there is no research to discuss the binding mechanism of the interaction between PHL and pepsin/trypsin.In addition,the solubility and stability of PHL limit its wide application in food.Therefore,the interaction model of PHL and pepsin/trypsin was established,and the binding mode of PHL with pepsin/trypsin was comparatively analyzed at the molecular level.On this basis,the commonly used nano-carrier zein was selected to prepare nanoparticles with zein and gum arabic as carriers to encapsulate phlorizin,and the prepared nanoparticles were added to yogurt to prepare nutrient-fortified yogurt.The research contents and results of this project are as follows:（1）The binding mode of the interaction between PHL and pepsin and trypsin was predicted by molecular docking and molecular dynamics.The results of molecular docking showed that hydrogen bond,van der Waals force and electrostatic force were mainly involved in the binding of PHL with pepsin/trypsin,maintaining the spatial protein structure.Molecular dynamics simulation analysis showed that PHL could bind with pepsin/trypsin stably,and the binding had a slight impact on the secondary structure of pepsin/trypsin,but it would not destroy the main structure of protease.（2）The interaction mechanism between PHL and pepsin/trypsin was investigated by multispectral techniques such as fluorescence spectroscopy,time-resolved fluorescence,circular dichroism spectroscopy,fourier transform infrared spectroscopy and antioxidant experiments.The fluorescence spectroscopy calculations showed that the ground state complex between PHL and pepsin/trypsin has a medium affinity of 10⁵L mol^-1.Thermodynamic parameters indicated that the binding of PHL to pepsin/trypsin proceeded spontaneously,with electrostatic force,van der Waals force and hydrogen bonding as the main forces.Circular dichroism spectroscopy and synchronous fluorescence spectroscopy showed a slight change in the spatial conformation of the protein after the binding of PHL to pepsin/trypsin and a change in the microenvironment in which the tryptophan residues were located.Fourier transform infrared spectroscopy showed that after the combination of PHL and pepsin/trypsin,the proteinβ-sheet content decreases and the secondary structure changes slightly.The results of antioxidant assays showed that PHL bound to trypsin exhibited stronger antioxidant activity than PHL bound to pepsin.（3）Nanoparticles encapsulated in PHL were prepared by the antisolvent precipitation method.The encapsulation efficiency of the nanoparticles encapsulated PHL prepared under the acidic condition of pH 4.0 is the best encapsulation rate.The main forces for nanoparticle formation were hydrogen bonding and electrostatic forces.The nanoparticles of zein and gum arabic encapsulated PHL have better salt ion stability,thermal stability and storage stability compared to the nanoparticles of zein alone encapsulated PHL.（4）The fortified yogurt with nanoparticles encapsulated PHL was prepared.The physicochemical properties of the yogurt were measured and analyzed.The results of water-holding capacity and antioxidant experiments showed that adding encapsulated PHL nanoparticles improved the water-holding capacity and antioxidant activity of yogurt.