Study On The Gastrointestinal Digestion And Fermentation Mechanism Of Protein-AGEs Complex

Hazard mechanism of advanced glycation end products(AGEs)present in food is closely associated with the digestion of protein-AGEs,absorption of AGEs,metabolism of absorbed AGEs in vivo and fermentation of unabsorbed AGEs by gut flora.Digestion of protein-AGEs was chosen as the entry of this study,followed by investigation of the relationship of structural and digestive changes of glycated protein.Distribution AGEs in digests and the fermentation of AGEs in digests by colon flora were studied subsequently.The main results are as follows:(1)Study on the multiscale structure of protein-AGEs complexDigestibility of a protein-AGEs is determined by its multiscale structure.Therefore the multiple structures of glycated ?-CN,?-Lg and BSA which were glycated by glyoxal(GO),methylglyoxal(MGO)and diacetyl(DA)were analyzed.Methods including SDS-PAGE and mass spectrometry were used to identify the change in primary structure of glycated proteins.AGEs with different characteristics(UV absorption,fluorescent characteristics or crosslinked structure)was found to accumulate during the glycation along with the decrease of lysine(Lys)and arginine(Arg)residues.Free sulfydryl group was found to reaction with ?-dicarbonyl compounds,giving rise to cysteine-derived AGEs such as S-carboxymethyl-cysteine(CMC).The secondary structure of glycated ?-CN appeared unchanged,whereas the ?-helix of BSA and ?-sheet of ?-Lg were reduced during glycation,according to the data collected from circular dichroism spectrometry.Glycation showed unobvious effect on the aggregation of ?-CN.However,glycation was found to inhibit the amorphous aggregation of BSA by adjusting both covalent(disulfide bond)and non-covalent(surface hydrophobicity,steric hindrance)interaction,transforming rigid globular aggregates into aggregates with loose and branched structures.Glycation was evidenced to change the kinetics of amyloid-like aggregation of ?-Lg,with increase in fibrillation rate and decrease in fibrillation capacity.The thermal stability of glycated fibrils was improved by glycation.(2)Digestive change of protein-AGEs complexA in vitro digestion model was used to simulate gastrointestinal digestion,the relationship structure of protein-AGEs complex and their digestibility change was investigated in this section.SDS-PAGE results indicate strong resistance of crosslinked glycation structure(especially from DA-derived glycation)on the enzymatic hydrolysis of glycated ?-Lg and BSA.Non-crosslinked structures were found to located near the cleavage site of digested peptides based on sequencing results of peptides in digests using LC-ESI-MS/MS,which imply the minor hindrance of these non-crosslinked structures against the action of proteases compared with crosslinked ones.A larger decline(declined by 30-90%)in degree of hydrolysis were found in ?-Lg-and BSA-AGEs complex than in ?-CN-AGEs complex(declined by less than 30%)during both gastric and intestinal digestion,thus implying smaller influence of glycation on the digestibility of ?-CN.This result should be in relation to the loose and flexible structure of ?-CN which weaken the hindrance of glycation structures against the action of digestive proteases.(3)Distribution of AGEs in digests of protein-AGEs complexThe digests of GO-glycated ?-CN/?-Lg was separated into fractions with different molecular ranges(>20 kDa,4.5-20 kDa,1-4.5 kDa and <1 kDa)by size exclusion chromatography(SEC),followed by identifying the distribution of AGEs with different characteristics using ultraviolet spectroscopy(UV),fluorescent spectroscopy and Mass spectroscopy.No free CML was found in digests,but presented in peptides with 6-14 amino acids.CML present more in digests larger than 1 kDa than lysine.The resistance of fluorescent AGEs to enzymatic hydrolysis gradually increased during glycation,rendering fluorescent AGEs largely present in the digests larger than 20 kDa.These results suggests the resistance of GO-derived AGEs to digestive proteases which is native barrier to the absorption of protein-bound AGEs,thus may imply less hazard of protein-bound AGEs than free and peptide-bound AGEs.(4)Degradation of AGEs in digests of protein-AGEs complex by colon flora.Degradation of AGEs in digests by colon flora were studied in an in vitro fermentation.Fermentation of AGEs by gut flora in determined by both the molecular weight of the digests.AGEs with UV absorption at 320 nm were found to be decomposed to some extend;AGEs with fluorescent characteristics showed high stability;CML with relative content of 18%-65% can be degraded by gut flora,and CML present in digests with higher molecular weight(>10 kDa)showed higher stability than in digests with lower molecular weight(1-10 kDa,<1 kDa).In addition,composition of gut flora was found to be changed by the digests to different degree.Addition of digests with molecular weight higher than 10 kDa reduced the content of Weissella and bifidobacterium,which may pose a unfriendly effect on the gut health.This study should help to elucidate the health significance of dietary AGEs regarding digestion,absorption and gut health from new angle.