Preparation And Mechanism Analysis Of DPP-? Inhibitory Peptides Of Sheepskin Collagen

DPP-? inhibitors have become one of the main directions for the treatment of type II diabetes,and DPP-? inhibitory peptides extracted from food have also attracted attention due to their high safety and effectiveness.As a serine protease,DPP-? enzyme can specifically hydrolyze peptide containing Pro or Ala residues at the second NH₂ terminus.The collagen of sheepskin is rich in proline and has the potential to prepare DPP-? inhibitory peptides.And the waste of sheepskin in China is serious,resulting in environmental pressure.Therefore,this paper systematically studied the potential of sheepskin collagen as the precursor protein of DPP-? inhibitory peptides,the process of extracting DPP-? inhibitory peptides from sheepskin and the reason why collagen peptides has DPP-? inhibitory activity.The degradation of peptides during gastrointestinal digestion,and how to protect peptides from digestion were also studied.Firstly,the potential activity of sheepskin collagen was predicted by BIOPEP based on the amino acid sequence of sheepskin collagen.And the specific enzymatic hydrolysis sites of protease were combined to compare the possibility of DPP-? inhibitory peptides obtained from sheepskin by different simulating enzymatic hydrolysis.The results showed that sheepskin collagen has great potential for preparing DPP-? inhibitory peptides,and the use of Alcalase,Neutrase and Flavorzyme will result in a higher content of DPP-? inhibitory peptides.Secondly,the pretreatment process of extracting DPP-? inhibitory peptides was determined by protein extraction rate and degree of hydrolysis,and the best degreasing,alkaline hydrolysis and pyrolysis conditions were obtained.Furthermore,the degree of hydrolysis and molecular weight distribution were used to determine the best enzymatic hydrolysis process for preparing DPP-? inhibitory peptides.DPP-? inhibitory peptides were prepared with molecular weight below 1 k Da,which the content of peptides is between 39.83%and 80.4%.The DPP-? inhibitory activity of collagen peptides were measured by two evaluation methods in vitro and in Caco-2 cells.It was found that the DPP-? inhibitory activity of Flavorzyme hydrolyzed collagen peptides was lower than that of Alcalase and Neutrase,with IC50 of 49.52,26.02 and 20.66 mg/m L respectively.Flavorzyme have more restriction sites,which is not conducive to the preparation of DPP-? inhibitory activity.For further understanding the composition of the dominant DPP-? inhibitory activity peptides in collagen peptides,the different enzymatic collagen peptides were analyzed by LC-MS/MS,and the DPP-? inhibitory activity and half-life of peptides were predicted by BIOPEP and Expasy Prot Param software.The selected DPP-? inhibitory activity peptides were distributed between7 and 10 peptides according to the predicted DPP-? inhibitory activity greater than 0.5.According to the predicted DPP-? inhibitory activity,the dominant peptide segments were sorted from high to low,and four peptides with the highest predicted activity and the highest relative content were selected.Four selected peptides were synthesized to verify the activity and the mechanism of DPP-? inhibitory activity was studied by using enzyme kinetics and molecular docking.The results showed that GPAGPIGPV and GPAGPOGFPG showed high DPP-? inhibitory activity,with IC50 of 84.19 and 67.12?M,and the IC50 of GVVGLPG and GIOGVGPF are 178.51 and 125.42?M.GVVGLPG and GIOGVGPF did not bind to DPP-? active sites,and the effect of them with DPP-? was non-competitive inhibition.GPAGPIGPV and GPAGPOGFPG binding to the active site of DPP-?,and the effect of them with DPP-? belongs to competitive and non-competitive inhibition.In addition,Alcalase and Neutrase hydrolyzed collagen peptides were also prepared,and its DPP-? inhibitory activity in vitro was determined.Compared with the activity of single enzyme hydrolyzed collagen peptides,the Alcalase and Neutrase hydrolyzed collagen peptides showed lower activity.The changes of DPP-? inhibitory peptides during digestion were further investigated because bioactivity peptides are easily degraded during digestion.The Standard method was used to simulate gastrointestinal digestion,and the DPP-? inhibitory activity of collagen peptides after digestion were measured by two evaluation methods in vitro and in cells.The DPP-? inhibitory activity of collagen peptides were reduced(around 15%?50%).The decrease in oligopeptide content(around 18?30%)and the increase in free amino acid content(around30 per cent)after digestion may be the main cause of this result.The peptide components of ACP?NCP and FCP before and after digestion were analyzed by principal component analysis.It was found that the difference between collagen peptides would reduce after gastrointestinal digestion.Therefore,it is necessary to use enteric-coated capsules to protect the active peptides from degradation by digestive enzymes.The results showed that enteric-coated capsules have a significant protective effect on collagen peptides with DPP-? inhibitory activity.After protection,the activity of collagen peptides increased by 10%-20%.