| Diabetes (DM) is one of the fastest growing and the most extensive diseases worldwide, in recent years, the research of anti-diabetes drug-dipeptidyl peptidase IV (DPP-IV) inhibitor has attracted great interests of researchers. Looking for food protein-derived DPP-IV inhibitory peptides with natural, no side effects and diversed functional properties, is an important breakthrough to solve the side effects of DPP-IV inhibitor drugs. This research aimed to evaluate and compare the potential DPP-IV inhibitory sequences and its potential as the precursor between bovine and caprine milk casein (CN) by in silico analysis, then using protease hydrolysis to produce casein hydrolysate to verify its true DPP-IV inhibitory activity and composition differences between the two species. The casein hydrolysate was separated and fractionated by ultrafiltration and two-dimensional silica gel thin-layer chromatography (2D-TLC) and/or reversed phase high-performance liquid chromatography (RP-HPLC), then its containing high bioactive peptide was identified by liquid chromatograph/mass spectrometer(LC-MS). Moreover, the DPP-IV inhibition properties and mechanism of identified peptides were further investigated by the methods of enzyme inhibitory kinetics and molecular simulation technology, and the gastrointestinal digestion tolerability and absorption of DPP-IV inhibitory peptides, as well as their function to islet cell, were also discussed. The main research contents and results are as follows:(1) The potential DPP-IV inhibitory sequences and potential as the precursor of bovine and caprine milk casein were evaluated and compared by in silico analysis. All the four types of bovine and caprine milk caseins contain multiple DPP-Ⅳ inhibitory fragment, the DPP-IV inhibitory peptide occurrence (PC value) in caprine milk casein was k-CN> β-CN> αs1-CN> as2-CN, while that was β-CN> κ-CN> αs1-CN> αs2-CN in bovine casein; moreover, the DPP-IV inhibitory potential value of PI was κ-CN>β-CN> αs1-CN> αs2-CN for goat, while was κ-CN >αs1-CN> β-CN> αs2-CN for bovine. Considering both the four types of casein composition proportion and their individual PI value, the total DPP-IV inhibition potential (PIΦ) of goat milk casein, whether from the high (PIΦ,8.0410-6 μM-1g-1) or low (PIΦ,8.25 10-6 μM-1g-1) αs1CN genetic breed, has a weak better DPP-IV inhibition potential than bovine casein (PIΦ,7.7310-6 μM-1g-1). In addition, two efficient DPP-IV inhibitory sequences GPFPILV and HPINHR, selected from the peptide fragments released by trypsin in silico hydrolysis of goat milk casein, were found to have IC50 value of 163.7 and 452.2 μM, respectivly.(2) The true DPP-IV inhibitory activity and composition differences between bovine and caprine milk casein hydrolysate were verified. The bovine and caprine milk casein showed no DPP-Ⅳ inhibition, while remarkably different DPP-IV inhibitory activity was found in proteases hydrolysate, and DPP-IV inhibitory activity had no direct relationship with degree of hydrolysis. Caprine casein had better protease digestion characteristics than bovine casein, and most caprine casein proteases hydrolysates showed weakly but sigificantly higher DPP-IV inhibitory activity than that of bovine. Double enzyme digestion did not significantly increase the biological activity of goat casein hydrolysate. Trypsin treated hydrolysate showed the most efficient DPP-IV inhibitory activity, and goat casein hydrolysate has a lower/C50 value (0.801 mg/mL) than that of bovine (0.887 mg/mL). The two hydrolysates had obviously different molecular weight and hydrophobic properties composition, but the <5 kDa fraction has higher DPP-IV inhibitory activity than that of >5 kDa fraction for both sepecies, and common competition/noncompetition mixed DPP-IV inhibition mode was observed.(3) The casein hydrolysate was separated and fractionated by 2D-TLC and/or RP-HPLC, and its containing bioactive peptides were also identified by LC-MS/MS. A 2D-TLC (the first and second dimensional TLC developing solvent were chloroform respectively:methanol:25% ammonia (2:2:1) and n-butyl alcohol:acetic acid:water (4:1:1), respectively) and/or one-dimensional TLC (chloroform:methanol:25% ammonia (2:2:1) as developing solvent) combinated with RP-HPLC method were set up, which was suitable for the separation and analysis of bioactive components in the protein hydrolysate. Four new effective casein-derived DPP-IV inhibitory sequences MHQPPQPL, SPTVMFPPQSVL, INNQFLPYPY and YPVEPF were identified by LC-MS/MS, especially, INNQFLPYPY had an IC50 value of 40.08μM.(4) The DPP-IV inhibition properties and mechanism of identified peptides were investigated. Enzyme inhibition kinetics analysis showed that the identified food protein-derived DPP-IV inhibitory peptides behaved competitive, noncompetitive, uncompetitive and competitive/noncompetitive mixed inhibition manners. Uncompetitive or noncompetitive inhibitory peptides have good structure stability, which could be regarded as real DPP-IV inhibitor, while competitive or competitive/noncompetitive mixed inhibitory peptides contain a second Pro could be degradated with different degree. Molecular simulation data showed that these peptides could contact with different amino acids of DPP-IV enzyme mainly through hydrogen bonding and hydrophobic interaction. The DPP-IV inhibitory peptides also showed obvious interaction on inhibiting DPP-IV when exsiting together, which was related to the peptide concentration. In addition, pig DPP-IV showed significantly higher susceptibility than human DPP-IV for inhibitory peptides.(5) The gastrointestinal digestion stability and absorption of casein-derived DPP-IV inhibitory peptides, and their function to islet cell, were investigated. The bovine and caprine milk casein hydrolysate showed good stability to gastrointestinal simulated digestion, and their conntaining effective peptide MHQPPQPL, HPINHR and YPVEPF don’t possess cleavage sites of digestive enzymes in their sequences, while INNQFLPYPY and GPFPILV showed unstable digestive enzyme hydrolysis resistance. completely different intake capacity and absorption rate of the small intestinal epithelial cells for INNQFLPYPY, INNQFand MHQPPQPL were observed by confocal laser experiment. In addition, bovine and caprine milk casein hydrolysate had no significantly different proliferation effect on islet beta cell, while bovine casein hydrolysate showed significantly stronger insulin secretion promoting effect than the goat counterparts. |
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