Preparation Of Differential Peptide And Peptide Zinc Complexes And Their Absorption And Metabolism Mechanism Based On Caco-2 Cell Model

The absorption of zinc by the human body was not only dependent on adequate dietary intake,but also greatly influenced by the intestinal utilization rate.The absorptive capacity of Pep T1 carrier to different active peptides was different.Generally,the affinity of negatively charged peptides of Pep T1 carrier is better than that of positively charged peptides.For hydrophobic peptides,the stronger the hydrophobicity,the easier the absorption of Pep T1carrier.This is also an important factor affecting the absorption of peptides and their zinc complexes.Further elucidation of the influence of these factors on intestinal epithelial absorption of zinc remains an important research topic.Therefore,in this thesis,differentiated peptides with different charges and hydrophobicity were separated from soybean meal,and coordinated with Zn²⁺to form Zn-peptide complexes,respectively.The molecular structure and physicochemical properties of different peptides and their complexes were analysed.The absorption and metabolism mechanism of peptides and their complexes were explored through the Caco-2 cell model.1.Using soybean meal as raw material,LMWP with high degree of hydrolysis and low molecular weight was prepared by pretreatment,double enzyme enzymolysis and membrane separation and coupling.The weak hydrophobic peptide(WHP),strong hydrophobic peptide(SHP),positively charged peptide(PCP)and negatively charged peptide(NCP)were obtained by differential separation and purification using DA201-C macroporous resin and DEAE anion exchange column,respectively.The molecular weight distribution of these five peptides were analyzed,and it was found that most of the peptides were distributed between 3.5-5.0 k Da.2.After separation and purification,five peptides were coordinated with Zn²⁺to prepare Zn-peptide complex,respectively.The zinc coordination ability of positively charged peptide(PCP)(124.89±1.98mg/g)was higher than that of negatively charged peptide(NCP)(106.88±0.94mg/g).The zinc coordination ability of strong hydrophobic peptides(SHP)(89.65±2.03mg/g)was higher than that of weak hydrophobic peptides(WHP)(79.10±1.51mg/g),while that of low molecular weight peptides was 102.95±1.72mg/g.3.Study on the structure characterization and binding mechanism of Zn-peptide complexes:X-ray diffraction and spectral analysis showed that Zn²⁺could coordinate with the active groups(-COOH,-OH,-NH)of peptides to form Zn-peptide complexes.The results of SEM-EDS showed that the Zn-peptide complex had a dense granular structure.The thermogravimetric analysis showed that the Zn-peptide complexes had good thermal stability,and the weight loss between 120-400?was much less than that of peptides.In addition,both the Zn-peptide complexes and Zn SO₄ had good solubility under acidic conditions,but the zinc solubility of the Zn-peptide complexes(both higher than 40%)were significantly higher than that of Zn SO₄(7.54±1.16%)at intestinal pH 7.5.Similarly,in simulated intestinal digestion,the zinc release rate of Zn SO₄ was significantly lower than that of Zn-peptide complexes,while in simulated gastric digestion,Zn-peptide complexes had a certain ability to resist gastrointestinal digestion.These results indicated that the Zn-peptide complexes had better stability and higher bioavailability than Zn SO₄ in intestinal environment.4.Caco-2 cell model was established.The integrity and permeability of the Caco-2 cell model were evaluated by Caco-2 cell morphology,TEER measurement,alkaline phosphatase(AKP)activity detection,luciferin sodium transmittance and apparent permeability coefficient.The results showed that the Caco-2 cells were cultured for 21 days.The cells were closely bound,the resistance value was>300?·cm2,the AKP ratio of AP/BL was 5.60±0.22,the apparent permeability of luciferin was within the reference value,and the transmittance was only 4.56±0.17%.The above related parameters and indicators indicated that the Caco-2 cell model had been successfully established in this thesis,which could be used for the transport and absorption experiments of peptides and their Zn-peptide complexes.5.First,the concentration of Zn²⁺with the highest survival rate of Caco-2 cells was determined to be 50umol/L.At this concentration,the zinc transport and absorption efficiency of the Zn-peptide complexes and Zn SO₄ in Caco-2 cells were compared,and it was found that the Zn-peptide complexes had a higher absorption rate.The expression levels of related genes and enzyme lines in Caco-2 cells were further determined.It was found that the expression levels of ZIP4 mRNA and Zn T1 mRNA were proportional to the zinc concentration in a certain range of zinc concentration.The expression level of Pep T1 mRNA was mainly affected by peptides.Under the stimulation of peptides,the expression level of Pep T1 mRNA was up-regulated.The activity of carboxypeptidase A and Cu-Zn SOD were affected by the concentration of zinc,and Zn²⁺could catalyze or activate the activity of the enzymes.The results of gene and enzyme expression levels showed that the Zn-peptide complex groups were higher than other groups.The above results showed that Zn-NCP had the highest absorption efficiency and catalytic effect of gene and enzyme system.