Study On The Structural Characterization Of Egg White Peptide Chelated Calcium And Its Pathways Of Promoting Calcium Absorption

Calcium is known as an important mineral for human body to maintain the health of human bones.Bone loss,which leads to metabolic bone diseases such as rickets and osteoporosis,has been proven to be mainly associated with insufficient calcium absorption.Related studies have shown that the amount of calcium absorbed by the body depends on the amount of soluble calcium in the proximal jejunum and duodenum.The solubility of calcium can be diminished due to the precipitation of calcium ions with antagonists such as cellulose,phytates and oxalate in the intestine,which inhibits the effective absorption of calcium.So it has raised concerns about developing new calcium supplements.Therefore the concerns on developing new calcium supplements and enhancing calcium solubility and absorption effects from food substances?particularly peptides?have been widely aroused.Binding calcium with peptides to prevent calcium precipitation can effectively increase the absorption of calcium in the body.In this study,a novel peptide from egg white,Asp-His-Thr-Lys-Glu?DHTKE?,contains specific amino acids associated with calcium binding.Egg white peptide chelated calcium?DHTKE-calcium?was prepared from DHTKE peptide,and investigated their structural characteristics.At the individual cell level,the fluorescence intensity inside individual cell was monitored in real time through Leica SP 8 laser confocal microscope,which could reflect the calcium-promoting effect of DHTKE-calcium chelate through the brush border membrane.At the group cell level,the calcium absorption characteristics of DHTKE-calcium chelate and its gastrointestinal digestive mixture were studied through Caco-2 cell monolayer model.Finally,different pathways promoters or inhibitors were used to treat Caco-2 cell monolayer to study the highly efficient calcium-promoting absorption pathways of DHTKE-calcium chelate.It provided a theoretical basis for the structural characterization,absorption characteristics and absorption pathways of potential calcium supplement.The results were as follows:1.Study on the structural characterization of DHTKE peptide and DHTKE-calcium chelate.By means of spectroscopy,spectroscopy and thermodynamics,the binding mode of egg white peptide and calcium ions were discussed,as well as the structural properties of DHTKE peptide and DHTKE-calcium chelate.The results showed that the calcium-binding site of the DHTKE might correspond to the carboxyl oxygen and amino nitrogen atoms of Glu and Asp together with the imidazole group of His.DHTKE peptide chelate could spontaneously bind calcium with a 1:1 stoichiometry.Therefore,DHTKE had high calcium ion binding activity.2.Study on establishment of Caco-2 cell monolayer model and its integrity evaluation.During the establishment of Caco-2 cell monolayer model,three methods were used to evaluate the integrity of cell membrane,namely,the transepithelial electrical resistance variation,laser confocal microscopy of Caco-2 cell monolayer,and the polarization of cell monolayers.When the cell monolayer was cultured at day 14,indicating that the cell membrane of Caco-2 has been highly polarized and differentiated into a complete monolayer membrane structure.Accurately,the results above indicated that the establishment of cell monolayer model was successful,which was similar to the intestinal epithelial absorption system in human body.3.Study on promoting calcium transport of DHTKE peptide and its gastrointestinal digestive mixture in Caco-2 cell monolayer.At the individual cell level,the fluorescence intensity inside individual cell was monitored in real time through Leica SP 8 laser confocal microscope,which could reflect the calcium-promoting effect of DHTKE-calcium chelate through the brush border membrane.At the group cell level,the calcium absorption characteristics of DHTKE-calcium chelate and its gastrointestinal digestive mixture were studied by means of Caco-2 cell monolayer model.The results showed that DHTKE-calcium chelate significantly increased the intracellular calcium ion content when compared to CaCl₂ control,and the effect of DHTKE peptide was more significant.In addition,DHTKE-calcium chelate had a certain structural stability in the intestinal digestinal process,and there was no significant difference between DHTKE-calcium chelate and its gastrointestinal digestive mixture in promoting calcium absorption?P>0.05?,indicating that digestion didn't affect the calcium absorption of DHTKE-calcium chelate.4.Study on calcium-promoting absorption pathways of DHTKE-calcium chelate through human intestinal cells.Cell monolayer models were treated with promoters or inhibitors of different pathways?transcytosis,TRPV6 calcium channel,Ca_v1.3 calcium channel and paracellular pathway?to study the calcium-promoting absorption pathways of DHTKE-calcium chelate through human intestinal cells.Results above indicated that DHTKE-calcium chelate promoted calcium absorption through multiple pathways,involving the Ca_v1.3 calcium channel,the endocytic pathway and the paracellular pathway.