Intestinal Absorption Mechanism Of Myofibrillar Protein-bound N~?-(Carboxymethyl) Lysine

Advanced glycationendproducts(AGEs)are mainly a series of complex mixtures of free amino groups of proteins(or amino acids)and carbonyl groups of reducing sugars(such as glucose,fructose,etc.)through the nonenzymatic browning pathway.Dietary AGE is an important environmental factor of body AGEs pool,which is closely related to the pathogenesis of some chronic diseases.In particular,the level of AGEs in meat products is the highest.This study used myofibrillar protein-bound N?-(Carboxymethyl)lysine(MP-bound CML),a representative marker of AGEs in meat products,as the research object.Myofibrillar protein(MP)was initially extracted from pork tenderloin.MP-bound CMLwas thenprepared by chemical synthesis.This study aimed to explore the changes in molecular morphology of MP-bound CML in the gastrointestinal digestion in vitro and to analyze the level of CML in hydrolysates of different molecular weight ranges after digestion.Caco-2 cell model was established to evaluate the absorption rate and absorption mechanism of MP-bound CML hydrolysates in different molecular weight ranges.This will provide a theoretical basis for scientific evaluation of the intestinal absorbability of protein-bound CML in food.The main results are as follows:Firstly,the paper investigatedthe changes of MP-bound CML in the process of simulating the digestion of the gastrointestinal system in vitro using fluorescence microscopy,electrophoresis and particle size analyzer.Results showed that the particle size of MP-bound CML gradually decreasedwiththe increase of digestion time.When the digestion time was 360 min,the particle size of MP-bound CML hydrolysate was in the range of 100 nm-1000 nm,and the molecular weight wasmainly below 17 k Da.Furthermore,ultrafiltration was used to classify the hydrolysate after digestion of gastrointestinal tract.The content of CML in different components was determined by HPLC-QQQ-MS / MS method.The results showed that the proportion of bound CML in the MP-bound CML hydrolysate with a molecular weight less than 5 k Da was 68.5%,and the proportion of bound CML with a molecular weight of less than 1 k Da was 8.9%.Secondly,the paper exploredthe intestinal absorption characteristics of MP-bound CML hydrolysates in different molecular weight ranges by establishing a Caco-2 cell monolayer membrane model.Results indicatedthat the optimal transport concentration of MP-bound CML hydrolysate in different molecular weight ranges was 600 ?g/m L.Three indexes of microstructure,transmembrane resistance,and sodium fluorescein flux through the membrane were used to evaluate the compactness and integrity of Caco-2 cell model,and the data proved that the Caco-2 cell modelsatisfiedthe requirements of membrane transport research.CML absorption rate of the component with molecular weight less than 1 k Da was 74.08 ± 8.93%,which was considered as an easily absorbed component.While CML absorption rates of components with molecular weight bewteen 1 k Da and 3 k Da as well as molecular weight bewteen 3 k Da and 5 k Da were 42.04±4.22% and 50.21±3.57%,respectively,which were regarded as relatively easy to absorb components.CML absorption rate of component with molecular weight greater than 5 k Da was 3.24 ± 1.71%,which was consideredextremely difficult to absorb.Finally,the paper clarified the absorption mechanism of MP-bound CML hydrolysates in different molecular weight ranges.Results indicated that CML components with molecular weight less than 1 k Da was absorbed by cell bypass without active efflux.Meanwhile,CML components with molecular weight bewteen 1 k Da and 3 k Da was absorbed by cell bypass and endocytosis without active efflux.Furthermore,CML component with molecular weight bewteen 3 k Da and 5 k Da was absorbed by Pep T1-mediated active transport without active efflux.Whereas CML components with molecular weight greater than 5 k Da was transported by active efflux.Based onc above data,the difficulty and mechanism of absorption and utilization of hydrolysates of different molecular weight components were different.There existed active efflux in CML components with high molecular weight,which was hard to be absorbed during the intestinal absorption process.This finding suggests that the risk of MP-bound CML being absorbed into the intestine is low.This paper will provide a certain theoretical basis for exploring the digestion and absorption of protein-bound AGEs in foods.