Glycation sites and bioactivity of lactose-glycated caseinate hydrolysate in lipopolysaccharide-injured IEC-6 cells

摘要

During the thermal processing of milk, Maillardreactions occur between proteins and lactose to generateglycated proteins. In this study, a lactose-glycatedcaseinate was hydrolyzed by trypsin. The obtainedglycated caseinate (GCN) hydrolysate had a lactosecontent of 10.8 g/kg of protein. We identified its glycationsites and then assessed it for its protective effectagainst lipopolysaccharide-induced barrier injury usinga rat intestinal epithelial cell line (IEC-6 cells) as acell model and unglycated caseinate (CN) hydrolysateas a reference. Results from our liquid chromatography–mass spectrometry analysis of the GCN hydrolysateverified that lactose glycation occurred at the Lysresidues in 3 casein components (αS1-casein, β-casein,and κ-casein), and this resulted in the formation of5 peptides with the following amino acid sequences:EMPFPKYPKYPVEPF, HIQKEDVPSE, GSENSEKTTMPL,NQDKTEIPT, and EGIHAQQKEPM. Theresults from cell experiments showed that the 2 hydrolysatescould promote cell growth and decrease lactatedehydrogenase release in the lipopolysaccharide-injuredcells; more importantly, they could partially protectthe damaged barrier function of the cells by increasingtrans-epithelial electrical resistance, decreasingepithelial permeability, and upregulating the expressionof the 3 tight junction proteins zonula occludens-1,occludin, and claudin-1. However, compared with CNhydrolysate, GCN hydrolysate showed lower efficacy inprotecting against cellular barrier dysfunction. We proposethat the different chemical characteristics of theCN hydrolysate and the GCN hydrolysate (i.e., aminoacid loss and lactose conjugation) contributed to thelower barrier-protective efficacy of the GCN hydrolysate.During dairy processing, protein glycation of theMaillard type might have a non-negligible, unfavorableeffect on dairy proteins, in view of the resulting proteinglycation we found and the critical function of proteinsfor maintaining the integrity of the intestinal barrier.