N-ε-fructosyllysine and N-ε-carboxymethyllysine, but not lysinoalanine, are available for absorption after simulated gastrointestinal digestion.

Food processing leads to a variety of chemical modifications of amino acids in food proteins. Recent studies have shown that some modified amino acids resulting from glycation reactions can pass the intestinal barrier when they are bound in dipeptides. In this study, we investigated as to what extent modified amino acids are released from post-translationally modified casein during simulated gastrointestinal digestion. Casein was enriched with N-ε-fructoselysine, N-ε-carboxymethyllysine, and lysinoalanine, in different degrees of modification. The casein samples were subjected to a two-step proteolysis procedure, simulating gastrointestinal digestion. The digestibility of modified casein as measured by analytical size-exclusion chromatography (SEC) decreased with increasing degree of modification especially after enrichment of fructoselysine and lysinoalanine. Semi-preparative SEC of digested casein samples revealed that fructoselysine and carboxymethyllysine are released bound in peptides smaller than 1,000 Da, which is comparable to native amino acids. The glycation compounds should, therefore, be available for absorption. Lysinoalanine as a crosslinking amino acid, however, is mostly released into longer peptides of at least 30-40 amino acids which should strongly impair its absorption availability.

An esterase from the basidiomycete Pleurotus sapidus hydrolyzes feruloylated saccharides.

Investigating the secretion of esterases by the basidiomycetous fungus Pleurotus sapidus in a Tween 80-rich nutrient medium, an enzyme was discovered that hydrolyzed the ester bond of feruloylated saccharides. The enzyme was purified by ion exchange and size exclusion chromatography. Polyacrylamide gel electrophoresis analysis showed a monomeric protein of about 55 kDa. The complete coding sequence with an open reading frame of 1,665 bp encoded a protein (Est1) consisting of 554 amino acids. The enzyme showed no significant homology to any published feruloyl esterase sequences, but possessed putative conserved domains of the lipase/esterase superfamily. Substrate specificity studies classified the new enzyme as type-A feruloyl esterase, hydrolyzing methyl ferulate, methyl sinapate, and methyl p-coumarate but no methyl caffeate. The enzyme had a pH optimum of 6 and a temperature optimum at 50 °C. Ferulic acid was efficiently released from ferulated saccharides, and the feruloyl esterase exhibited moderate stability in biphasic systems (50 % toluene or tert-butylmethyl ether).

Transport of free and peptide-bound glycated amino acids: synthesis, transepithelial flux at Caco-2 cell monolayers, and interaction with apical membrane transport proteins.

In glycation reactions, the side chains of protein-bound nucleophilic amino acids such as lysine and arginine are post-translationally modified to a variety of derivatives also known as Maillard reaction products (MRPs). Considerable amounts of MRPs are taken up in food. Here we have studied the interactions of free and dipeptide-bound MRPs with intestinal transport systems. Free and dipeptide-bound derivatives of N(6)-(1-fructosyl)lysine (FL), N(6)-(carboxymethyl)lysine (CML), N(6)-(1-carboxyethyl)lysine (CEL), formyline, argpyrimidine, and methylglyoxal-derived hydroimidazolone 1 (MG-H1) were synthesized. The inhibition of L-[(3)H]lysine and [(14) C]glycylsarcosine uptakes was measured in Caco-2 cells which express the H(+)/peptide transporter PEPT1 and lysine transport system(s). Glycated amino acids always displayed lower affinities than their unmodified analogues towards the L-[(3)H]lysine transporter(s). In contrast, all glycated dipeptides except Ala-FL were medium- to high-affinity inhibitors of [(14)C]Gly-Sar uptake. The transepithelial flux of the derivatives across Caco-2 cell monolayers was determined. Free amino acids and intact peptides derived from CML and CEL were translocated to very small extents. Application of peptide-bound MRPs, however, led to elevation (up to 80-fold) of the net flux and intracellular accumulation of glycated amino acids, which were hydrolyzed from the dipeptides inside the cells. We conclude 1) that free MRPs are not substrates for the intestinal lysine transporter(s), and 2) that dietary MRPs are absorbed into intestinal cells in the form of dipeptides, most likely by the peptide transporter PEPT1. After hydrolysis, hydrophobic glycated amino acids such as pyrraline, formyline, maltosine, and argpyrimidine undergo basolateral efflux, most likely by simple diffusion down their concentration gradients.