Study On The Identification And The Antioxidant Mechanism Of Antioxidant Peptides In Xuanwei Ham

Reactive oxygen species(ROS)are signaling molecules produced in vivo and play important roles in regulating mitochondrial respiration and enzymatic reactions.However,with the reduction of physiological function and the impact of the external environment including UV,chemical pollution,radiation and air pollution and other stimulation,biological reactive oxygen radicals will gradually accumulate in cell along with the generation of oxidative stress reaction.In order to alleviate the physiological damage caused by oxidative stress,supplementation of antioxidant ingredients is of great significance to maintain physiological health.In plants,animals,microorganisms and other natural raw materials,the bioactive peptides were tested to be existed and their activity were closely related to the protein source,structure and amino acid composition of the biopeptide.Dry-cured ham,as a kind of meat product with a long history of processing,has been widely accepted by consumers for its flavor and product quality.Duing the production of dry-cured ham,the proteins in meat were degraded with the activity of endogenous enzyme along with the generation of bioactive peptides.In order to explain the functional properties of dry-cured ham,the current research focsed on the study of protein degradation,antioxidant properties and absorption characteristics of active peptides in dry-cured ham.Based on the methods of proteomic analysis,peptide purification separation,cell electrochemical testing and cell culture,the current study gave further illustrations on the antioxidant pathways,digestive characteristics,transmembrane transport pathway and the regularity of antioxidation mechanisms of Xuanwei ham antioxidant peptides.The specific research contents and results are as follows:1.The formation and evaluation of antioxidant peptides in dry-cured Xuanwei hamChosing Xuanwei and Jinhua ham as material,the bioactive peptides were extracted in ham muscle.Compared with the activity of glutathione(GSH),the antioxidant activity of bioactive peptides was tested on scavenging effect on the 1,1-dipheny 1-2-trinitrobenzene hydrazine(DPPH)free radical,superoxide anion radical(O2-·),ABTS radical as well as the oxygen free radicals absorptive capacity(ORAC).At the same time,the LC-ESI-Q-TOF/MS/MS method was used to identify the peptide components and analyze the relationship between the degraded peptide components and the mother proteins.The results showed that DPPH and O2-· scavenging activity of polypeptides in Xuanwei and Jinhua ham all improved in the increased concentration.The ORAC(949.97 mol TE/g)and ABTS(148.97 mol TE/g)of Xuanwei ham peptides were higher than Jinhua ham.There were 243 and 213 bioactive peptides identified in Xuanwei and Jinhua respectively,where the source proteins mainly derived from myosin,troponin and actin.Among them,the two kinds of ham had the same polypeptide sequence,where the amino and carboxyl end of some polypeptide sequences had similar peptide chain indicating that aminopeptidase and carboxylpeptidase play an important role in the production of polypeptides.2.Isolation,purification and identification of antioxidant peptides in dry-cured Xuanwei hamIn this chapter,Xuanwei ham was selected as the raw material to purify the antioxidant peptides.Firstly,polypeptide components in the ham were extracted.Then,G-25 gel chromatography column,ion exchange column,high performance liquid chromatography and other technologies were used to purify polypeptides in Xuanwei ham.Meanwhile,DPPH and O2-.activities were used to detect the function of the purified polypeptide,so as to select the fragment with high antioxidant activity.After G-25 purification,the DPPH and O2-.scavenging activities of component C were all higher than those of other components.After ion exchange column chromatography,the antioxidant activity of component C5 increased significantly.After the preparative liquid phase separation,the peak antioxidant activity of C5-7 was the highest relative to other peak fragments.Finally,C5-7 was analyzed by liquid phase mass spectrometry,and the obtained peptide fragment was Asp-Leu-Glu-Glu(DLEE).The peptide sequence was synthesized and its activity was verified by DPPH free radical scavenging activity.The synthetic sequence had the same DPPH free radical scavenging activity as the purified peptide indicating that the DLEE plays a major role in antioxidant function.3.Evaluation of antioxidant activity of DLEE by cell electrochemical methodBased on the electrochemical detection by pulse voltammetry,a cell electrochemical platform was constructed by combining Caco-2 cells with modified electrodes,and the antioxidant activity of DLEE was evaluated with the help of cell electrodes.Firstly,platinum nanoparticles and silver nanowires were selected as electrode modification materials.Meanwhile,sodium alginate,graphene and DMEM cell culture medium were mixed to prepare cell gel.Subsequently,Caco-2 cells were incubated with peptides and treated with H2O2 oxidative damage.Finally,Caco-2 cells incubated by DLEE and treated with oxidative damage were fixed on electrochemical electrodes to detect the strength of electrochemical redox signal.Therefore,the antioxidant effect of DLEE in Caco-2 cells was verified.The results showed that the electrochemical cell sensor had a good sensitivity to detect H2O2 concentration in Caco-2 cells.Within the range of 0.2-2 ?M,there was a linear relationship between H2O2 concentration and current value,and the lowest detection line was 0.12 ?M.Meanwhile,the sensor was applied to detect the antioxidant capacity of the antioxidant peptide DLEE in Xuanwei ham.The antioxidant value of DLEE was obtained as 88.17%.Therefore,the cell sensor is feasible to evaluate the antioxidant capacity of polypeptides.4.The digestion and transmembrane absorption of DLEE in Caco-2 cellsIn this chapter,pepsin and trypsin were selected to simulate the digestion process in vitro and the digestive stability of DLEE was analyzed by liquid phase assay.Firstly,Caco-2 cells were selected to construct the intestinal epithelial cell model to explore the transport pathway of DLEE.The results showed that DLEE had no enzymatic degradation after the treatment by digestive enzymes.After 21 days,the Caco-2 cells met the experiment requirements in terms of monolayers polarity and alkaline phosphatase activity.The transmembrane transport of DLEE was affected by different incubation time and concentration.At the brush edge of Caco-2 cell monolayer membrane,DLEE could keep stable and the concentration showed to be increased gradually in base side.Finally,Gly-Pro,wormanpenicillin,sodium deoxycholic acid,sodium azide and cytochalasin D were added to the top side of Caco-2 cells.By adding inhibitors and promoters of different transport molecule along with the detecting of DLEE,we found that cytochalasin D,the bypass transport promoter,was the main factor affecting the transport of DLEE.The TEER value was significantly decreased after the transport of DLEE in Caco-2 cell.In addition,DLEE could decrease the expression of Occludin and increase transmembrane transport.5.The cytoprotection and antioxidant mechanism of DLEE in Caco-2 cellsIn this chapter,Caco-2 cells were used as the carrier and DLEE incubation treatment was combined with H2O2 oxidative damage which was aimed to explore the mechanism of antioxidant peptides on cytoprotection activity.After the DLEE incubation,oxidative damage was carried out with the treatment of H2O2,where the intracellular ROS production and total antioxidant value were observed.Cell proteins were extracted and the catalase(CAT),glutathione transferase(GR)and glutathione peroxidase(GSH-px)activities were detected.Furthermore,the expressions of Nrf2 and Keapl in the antioxidant pathway were further analyzed with Western Blot.Finally,the effects of DLEE on apoptosis were analyzed by loss cytometry.The results showed that H2O2 oxidative damage treatment could induce the increase of intracellular ROS production and inhibit the activity of antioxidant enzymes.DLEE incubation can alleviate oxidation-induced ROS production and restore antioxidant enzyme activity.Combined with the expression of antioxidant signaling pathway proteins Nrf2 and Keapl,the oxidative damage was shown to inhibit Nrf2/Keapl pathway,while DLEE could help alleviate the expression of Nrf2 protein in this pathway along with regulating the activity of downstream antioxidant enzymes.