Preparation And Activity Study Of Bioactive Peptides From Pecan Cake

Pecan cake,a by-product of pecan processing,has not been fully developed and utilized.It contains proteins with high nutritional value that can be used as a potential source of bioactive peptides.In this study,the compound protease from the four common proteases was selected as the best protease,and the best hydrolysis was determined via response surface optimization.The optimal hydrolysis conditions were as follows:55℃,pH 7.7,3900 U/g protease concentration,and 2 h.Under these conditions,peptide yield and total antioxidant capacity were 34.13±0.2%and 1.06±0.08 mmol/g,respectively.The peptides showed desirable DPPH(IC50:0.249 mg/mL)and ABTS(IC50:0.362 mg/mL)radical scavenging capacities,which were found to be related to their amino acid compositions,molecular weights,and structures.The molecular weight of the peptides obtained from the pecan cake protein hydrolyzed by the compound protease was<3 kDa,with their contents reaching 94.03±0.41%.HPLC analysis revealed that the content of hydrophobic amino acids in the peptides was relatively high.Fourier transform infrared spectroscopy and X-ray diffraction analysis showed that hydrolysis gradually changed the structure of the proteins from ordered to disordered,thereby exposing more antioxidant groups.α-helix and β-turn angles decreased from 29.49%and 24.9%to 20.31%and 15.16%,respectively.β-sheet,β-antiparallel,and random coil increased from 24.23%,7.72%,and 13.66%to 33.45%,9.30%,and 21.79%,respectively.In the current study,ultrafiltration and gel filtration chromatography are used to obtain peptides with strong antioxidant activities from hydrolysates of Chinese pecan cake.The substances with molecular weight(MW)<3 kDa have the highest antioxidant capacity,and they were identified through Gel filtration chromatography and LC-MS/MS.Six peptides have been identified,among those,VYGYADK and VLFSNY show the strongest antioxidant capacities.The crucial antioxidative interaction site of VYGYADK action on DPPH and ABTS was proved to be Tyr.The results of molecular docking indicate that VYGYADK and VLFSNY can combine with key amino acid residues of a transcription factor Kelch like ECH-associated protein 1(Keapl).The assay of cell viability reflected significant cytoprotective effects against H2O2-induced cellular oxidative damage(500-2000 μM).Meanwhile,VYGYADK and VLFSNY significantly reduced intracellular ROS generation,along with increased the activity of SOD and CAT.This study reveal the antioxidant mechanisms of VYGYADK and VLFSNY by modulating cellular antioxidation through Keap1-Nrf2-ARE signaling pathway.The α-glucosidase inhibitory activity assay was performed on the three fractions>10 kDa,3-10 kDa and<3 kDa obtained by ultrafiltration,and the results showed that the<3 kDa fraction showed the highest inhibitory activity at 3 mg/mL The inhibition rate reached 47.90%.Peptides were identified from fractions<3 kDa by Nano LC-MS/MS technology,and 31 peptides with scores above 0.8 were predicted using the Peptide Ranker website.Molecular docking of amylase was carried out,and four peptides with possible hypoglycemic activity were screened out with the lowest binding energy as the index,namely PWLYFI,HAFFPL,WIFWVGP and QFPEW.PWLYFI,WIFWVGP and QFPEW all showed certain α-glucosidase and α-amylase inhibitory activities.In terms of inhibition rate,the inhibitory activities of α-glucosidase and α-amylase were PWLYFI>WIFWVGP>QFPEW.Among them,PWLYFI had the highest α-glucosidase andα-amylase inhibition rates,reaching 57.17±6.32% and 51.71±2.76% at 3 mg/mL.