Interaction Mechanism Of Phlorizin And β-Lactoglobulin Under Thermal Processing And Its Effect On Intestinal Mucosal Barrier

In recent years,functional dairy products have become popular with consumers,and by adding plant-based active substances,they not only satisfy the rich nutrition and good flavour of dairy products,but also benefit gastrointestinal digestion and absorption.Plant-based ingredients contain a large number of naturally occurring polyphenols that may coexist with milk proteins and can interact with each other during food processing.Phlorizin(PZ),a major component of apple polyphenols,has yet to be investigated for its interaction with Beta-Lactoglobulin(β-LG),a major allergen in milk,and its effect on the intestinal mucosal barrier.These studies will contribute to a better understanding of the interactions between food components and their physiological activity in gastrointestinal digestion.Firstly,the interaction between PZ and β-LG was simulated by molecular docking,and then the interaction mechanism of PZ-β-LG and the effect on the secondary structure of β-LG under thermal processing at 60℃/20 min,80℃/20 min and 100℃/5 min were investigated by spectroscopic techniques.Then the INFOGEST program was applied to simulate the gastrointestinal digestion of PZβ-LG complex and followed by the analyzesis of its antigenicity,antioxidant capacity and digestive peptide profile;finally,the effect of the digests on expression and secretion of mucin MUC2 in LS174T cell were investigated.The main studies and results are as follows:(1)Exploration of the interaction mechanism between Phlorizin and β-LG under thermal processing.Molecular docking simulations of PZ binding to β-LG revealed that the binding site is within the hydrophobic cavity of the "Calyx structure" and may mask some β-LG antigenic epitopes;UV-Vis absorption spectroscopy showed that the PZ-β-LG interaction could form a more stable complex after thermal processing and affect the β-LG chromogenic environment.And the microenvironment around amino acids.The fluorescence spectra showed that PZ could cause a static quenching of β-LG endogenous fluorescence,with hydrophobic forces dominating the formation of the complex and one stable binding site.Different thermal processing conditions caused changes in the main interaction forces between the two,with van der Waals and hydrogen bonding forces dominating at 60℃/20 min and 80℃/20 min,while electrostatic forces dominated at 100℃/5 min,but the type of quenching and the binding site remained unchanged.The results of circular dichroism showed that both thermal processing and Phlorizin altered the secondary structure of β-LG,with an increase in α-helix content,a decrease in irregular curl content leading to an increase in βLG stability.(2)Effect of thermal processing on the processing characteristics and antigenicity of PZ-β-LG.As the thermal processing temperature increased,the surface hydrophobicity and free sulfhydryl content of PZ-β-LG showed a decreasing trend,which in turn led to a decrease in emulsification and an increase in emulsion stability of PZ-β-LG,indicating that the Phlorizin had a masking effect on the hydrophobic group of β-LG.Indirect ELISA results showed that the antigenicity of β-LG was significantly reduced by the interaction of Phlorizin withβ-LG,but the difference in antigenicity between different thermal processing temperatures was not significant,indicating that Phlorizin masked the surface antigenic epitopes of the β-LG and that stable complex could be formed at room temperature.(3)In vitro simulation of the peptide profile and antigenicity of PZ-β-LG digestion under INFOGEST processing.In vitro simulations of gastric and gastrointestinal digestion of the different conjugates using the INFOGEST method were evaluated by the SDS-PAGE,Tricine-SDS-PAGE and degree of hydrolysis.The results showed that the addition of PZ promoted the hydrolysis of β-LG in gastrointestinal digestion and the hydrolysis of PZ-β-LG increased by 1.6%;the antigenicity of PZ-β-LG decreased and the DPPH radical scavenging ability increased by 1.2%after digestion,but the differences in the antigenicity and DPPH radical scavenging ability of β-LG before and after heating at 100℃/5 min were not significant.LC-MS/MS analysis of the four digests,β-LG,β-LG-100,PZ-βLG and PZ-β-LG-100,showed that the number and type of peptides produced differed,all being mainly 10-20 peptides,by producing two common hydrolysisresistant fragments LDAQSAPHLR and TPEVDDEALEK;β-LG-100 and PZ-βLG produced the largest and least,PZ-β-LG and PZ-β-LG-100 lacked peptide fragments KIIAEKTK and VLVLDTDYKK,and β-LG-100,PZ-β-LG and PZ-βLG-100 had an additional LIVTQTMKGLDIQK.100℃/5 min resulted in the alteration of the cleavage site of complexes in range of the amino acid sequence(45-57).(4)Phlorizin synergistically promoted the secretion of the MUC2 in LS174T cells.MTT assays determined that the optimal concentration of gastric and gastrointestinal digest was 5 μg/mL.The hydrolysis of PZ-β-LG promoted cell proliferative activity and that high concentrations of Phlorizin significantly inhibited cell activity.The staining method with Alisin Blue-Periodic Acid Schiff(AB-PAS)revealed that Phlorizin and PZ-β-LG increased the secretion of MUC2,but 100℃/5 min the difference was not significant.Protein immunoblotting(Western Blot)revealed that the complexes adding with Phlorizin promoted the expression and secretion of MUC2 protein in both intracellular and extracellular fluids after gastrointestinal hydrolysis.Meanwhile,RT-PCR results also showed an increase in the expression of cellular MUC2 mRNA.Phlorizin and PZ-β-LG digested peptide fragments may be involved in the protective effect of the intestinal mucosal barrier by enhancing the expression and secretion of MUC2 in LS174T cells.In this work,we found that Phlorizin could intercalate with β-LG at room temperature and the binding forces were affected by thermal processing.The intercalation resulted in reduced β-LG antigenicity,altered enzymatic sites and more benefit gastrointestinal digestion,as well as a synergistic promotion of MUC2 expression and secretion in LS174T cells.This study provides a theoretical support for polyphenol-protein interactions and hypoallergenic dairy systems.