Effect Of Glycation On The Formation And Intestinal Cell Absorption Of Bovine Serum Albumin Bound Quercetin Nanoparticle

In the present study,glycated bovine serum albumin(BSA)with different glycation degree was prepared via Maillard reaction by heating with two monosaccharide,xylose(Xyl)and galactose(Gal).The Xyl-or Gal-glycated BSAs were then combined with quercetin(QUE)(glycated BSA-QUE).At the same time,the mixture of BSA,Xyl/Gal and QUE was directly heating together(BSA-Xyl/Gal-QUE).Their structural characteristics,stability,antioxidant activity,effects on intestinal cell growth,and intestinal cellular uptake and absorption were investigated.The results are as follows:(1)When BSA was heated with monosaccharide(xylose,galactose,glucose,fructose)or disaccharide(lactose,maltose),a new band appeared in the high-molecular-weight area at 60℃ in SDS-PAGE,and its density was decreased at 70℃ and 80℃.The absorbance at 420 nm was increased when heated at 70℃ and 80℃.In three-dimensional fluorescence spectra,the intrinsic fluorescence excitation(λex)and emission(Xem)wavelengths exhibited blue-shift at different degrees,and the fluorescence intensity was decreased.The Maillard reaction products(MRPs)exhibited a new characteristic fluorescence peak,and its λex and Xem was mainly at 330～335 nm/400～405 nm,respectively.The wavelengths of MRPs exhibited red-shift with increasing of heating temperature and time.The characteristic fluorescence peak of MRPs could be used as an indicator of Maillard reaction progress,but only at low temperature of 50℃.(2)There were shifts of maximum wavelengths and decreases of fluorescence intensities for both intrinsic and ANS fluorescences;lower-wavenumber shifts of FT-IR amide Ⅰ,Ⅲ,and A bands;decrease of a-helix and increase of P-sheet percentages when BSA was heated.The hydrophobic groups became exposed and surface hydrophobicity was increased,resulting in BSA unfolding and aggregation.The size of BSA was increased,and its shape changed from spheroid particles to rod-like short chains,and its thermal stability was decreased after BSA was heated.Heat-induced structural changes and aggregation was inhibited after glycation.The glycated BSA kept as spheroid particles,and the thermal stability was increased.The combined quantity of saccharides and numbers of glycated residues were more for BSA-Xyl than BSA-Gal.The Xyl glycated residues were equally distributed in BSA peptide chain,while Gal glycated residues were located at spatially closed two ends.Glycation with Gal or Xyl restrained in similar degrees in the changes of fluorescence wavelengths,the amide I band and a-helix percentage.It is the glycation sites,but not glycation numbers plays an important role in restraining heat-induced protein structural change and aggregation.(3)The glycated residues number was decreased and their distribution became much more uneven when BSA was heated with Xyl/Gal and QUE.The bonding amount of QUE to BSA was decreased after BSA was heated or glycated,or adding of saccharides in BSA/QUE system.The bonding amount of QUE was decreased with the increasing of glycation degree.The fluorescence intensities for both intrinsic and ANS fluorescences of glycated BSA-QUE and BSA-Xyl/Gal-QUE were significantly decreased,and the maximum emission wavelengths shifted.The fluorescence changes were greater for BSA-Xyl/Gal-QUE than glycated BSA-QUE.The a-helix percentages were further decreased for BSA and glycated BSA after bonding with QUE.The decrease of a-helix percentages of BSA-QUE was restrained after glycation of BSA or addition of saccharides,and there was no significant difference between glycated BSA-QUE and BSA-Xyl/Gal-QUE.The particle size of BSA was increased after bonding with QUE,which was restrained after glycation of BSA or addition of saccharides.The length of chains for heated BSA became short after bonding with QUE,while glycation of BSA or addition of saccharides maintained spherical shape.The ζ-potential of BSA was decreased after heating or glycation or bonding with QUE,and the electrostatic repulsion among particles was increased,so as to keep the protein particles stable in solution.Glycation of BSA or addition of saccharides has no significant effect on the ζ-potential of BSA-QUE.The thermal stability of BSA and glycated BSA was improved after bonding with QUE,and glycation of BSA or addition of saccharides could improve the thermal stability of BSA-QUE.(4)BSA-QUE could inhibit the growth of Caco-2 cell by destroying the integrity of cell membrane,resulting in the increase of cell membrane permeability;and by promoting early apoptosis and blocking its DNA synthesis.After bonding with QUE,the ABTS+° and DPPH° free radical scavenging rates(RSR)of BSA were increased,and heating or adding saccharides had no significant effect on the RSR of BSA.The RSR of the BSA-Xyl/Gal-QUE were higher than those of glycated BSA-QUE.Both glycation of BSA or addition of saccharides in the BSA/QUE system could increase the absorption rate of BSA-QUE,but had no significant effect on the uptake efficiency.