Isolation,Purification And Identification Of Antioxidant Peptides From Corn Germ Meal And In Vitro Simulated Digestion And Absorption

The study was part of Changchun science and technology plan project--"Study on key technologies for production of series of foods for comprehensive utilization of corn germ meal"?14NK005?.In this study,corn germ meal was used as research object.Several components were obtained by enzymatic hydrolysis,ultrafiltration and G-25 gel chromatography.The antioxidant activities of each component were evaluated by DPPH radical scavenging activity,ABTS radical scavenging activity and Oxygen radical absorbance capacity?ORAC-FL?assay.The mass spectrometry technique was used to identify the amino acid sequence of the component with the best oxidative activity,and the corn germ antioxidant peptide was synthesized by solid phase synthesis.Then the activity of antioxidant peptides was evaluated by cellular antioxidant activity?CAA?assay,cell oxidative damage model,intracellular reactive oxygen species?ROS?levels and hydroxyl radical scavenging rate.Finally,the gastrointestinal fluid stability and absorption and transport properties of antioxidant peptides were explored by in vitro simulated gastrointestinal fluid digestion and establishment of Caco-2 cell absorption model,which provided a theoretical basis for its comprehensive utilization.The results of this research are as follows:?1?In the isolation,purification and identification of antioxidant peptides from corn germ meal,the five components?CGMH-1?>30 kDa?,CGMH-2?10-30 kDa?,CGMH-3?3-10 kDa?,CGMH-4?1-3 kDa?and CGMH-5?<1 kDa??were obtained by enzymatic hydrolysis and ultrafiltration.The antioxidant activities of these five components were evaluated by DPPH radical scavenging activity,ABTS radical scavenging activity and ORAC-FL assay.The results showed that CGMH-5 has the best antioxidant activity,and its DPPH radical scavenging rate,ABTS radical scavenging rate and ORAC value were 56.97±0.61%,88.18±0.67%and458.32±21.63?mol TE/g,respectively.Then CGMH-5 were purified by Sephadex G-25 gel chromatography,and four components F1,F2,F3 and F4 were obtained.The antioxidant activities of these four components were evaluated by the same methods.The results showed that F3 has the best antioxidant activity,and its DPPH radical scavenging rate,ABTS radical scavenging rate and ORAC value were 83.18±0.43%,equivalent to 0.53 mM BHT),86.88±0.35%?equivalent to 0.14 mM Trolox?and450.68±19.77?mol TE/g,respectively.Finally,F3 were identified by MS/MS.Three antioxidative peptides were obtained,and the amino acid sequences were Met-Gly-Gly-Asn?MGGN;377.42 Da?,Met-Asn-Asn?MNN;377.42 Da?and Met-Glu-Asn?MEN;392.43 Da?,respectively.?2?In the study of antioxidant activity evaluation of peptides,effects of antioxidant peptides on HepG2 cell growth were first detected by MTT assay.It was found that MGGN,MNN and MEN had neither toxic nor promoting proliferation on HepG2 cells in the range of 0.1-1.0 mM.Secondly,the results of CAA method showed that the quercetin equivalents of MGGN,MNN and MEN were 926.32?mol QE/100 mmol,1213.79?mol QE/100 mmol and 1083.08?mol QE/100 mmol,respectively,indicating that all three peptides have antioxidant activity,and MNN has the strongest antioxidant activity in cells.Thirdly,in the HepG2 cell oxidative damage model?modeling conditions:H₂O₂ concentration 600?M,treatment time 4 h?,MGGN,MNN,and MEN at concentrations of 0.4 mM,0.6 mM,and 0.8 mM all showed protective effects on HepG2 cells,and MNN has the best protection.Fourthly,the results of intracellular ROS scavenging capacity showed that MGGN,MNN and MEN at concentrations of 0.2 mM and 0.4 mM could significantly eliminate intracellular ROS?P<0.05?,and MNN has the strongest antioxidant activity.Finally,EPR was used to capture the number of·OH to visually detect the·OH clearance rate of antioxidant peptides.The results indicated that MNN had the strongest ability to scavenge·OH,with a value of 33.97±3.85%.?3?In the in vitro simulated digestion and absorption studies of MGGN and MNN,firstly,MGGN and MNN were found to have good gastrointestinal fluid stability by in vitro simulated gastrointestinal fluid digestion.Secondly,the integrity,differentiation polarity,permeability and cell morphology of the Caco-2 cell monolayer membrane model were evaluated.The results indicated that after 16 days of culturing Caco-2 cells,the value of TEER was stable at 700?·cm²;the alkaline phosphatase activity of the AP side was 11.10 times higher than that of the BL side;the Papp values of fluorescent yellow were between 0.1 and 0.7 after 60 min,120 min and 180 min incubation;the results of laser confocal microscopy showed that the cells were evenly distributed and compact.Therefore,Caco-2 cell monolayer model has been successfully established.Thirdly,using this model to study the absorption transport of MGGN and MNN,the results showed that if the concentration of the initial antioxidant peptide on the AP side was larger,the longer the incubation time,the more antioxidant peptides were absorbed into the BL side.And under the same transport conditions,the absorption content of MNN was greater than that of MGGN,indicating that the peptides with the same molecular weight and fewer amino acids have more absorption and translocation.Finally,the absorption mechanism of the antioxidant peptide MNN was further studied by adding the bypass absorption enhancer?cytochalasin D?,the endocytosis inhibitor?wortmannin?,the ATP production inhibitor?sodium azide?and the Pep T1 vector inhibitor?Gly-Sar?.The results showed that the content of MNN on the BL side of Transwell chamber containing Gly-Sar decreased from 0.87±0.03 mmol to 0.57±0.03 mmol,indicating that MNN relies on the Pep T1 vector for absorption and transport.