Stable Antioxidant Peptides Produced By Proteolysis And Their Functional Analysis

Because of the antioxidant efficiency is much higher than that of non-polypeptide antioxidants,polypeptide antioxidants have been widely used as antioxidant active ingredients.However,active polypeptides,like most active proteins,are often not stable enough in extreme applications such as high temperature.It has become the most important factor restricting its widespread use in cosmetics,health care products and pharmaceuticals.In this project,stable bioactive peptides capable of maintaining good antioxidant activity at high temperature were obtained from casein by enzymatic hydrolysis and high temperature screening,and the physiological functions and structural compositions of the peptides were analyzed.It provides reference for the study of the functional properties of antioxidant peptides and the study of temperature tolerance mechanism,and lays a foundation for the development and application of functional foods.The research results are as follows:1.Single factor and orthogonal experiments were used to optimize the hydrolysis conditions of casein by using superoxide anion radical,DPPH free radical and hydroxyl radical scavenging ability as detection indexes.The protein hydrolysate with higher antioxidant activity was obtained under the conditions of substrate concentration 10%,55°C,pH 6.5,reaction time 4 h,and enzyme dosage 35?L.2.The screening conditions for the high temperature resistant antioxidant polypeptide were determined.The hydrolysate with higher antioxidant activity obtained by optimizing the hydrolysis conditions was screened at 70,80,90,100°C.Based on the three kinds of free radical scavenging ability,it was determined that the hydrolysis mixture A₃B₂C₁D₃ treated at100°C for 25 min,could obtain a hydrolysate with good temperature tolerance and antioxidant activity.The product remained highly bioactive after treatment at 121°C for 5 h.3.Differential analysis of antioxidant peptide components after different high temperature treatments was carried out by reversed-phase high performance liquid chromatography.According to the peak retention time,the peak of the RSD%within 3%is defined as the characteristic peak of the same class of matter.A total of 37 characteristic peaks were obtained by 5 temperature treatments.Among them,the sample treated at 100°C and121°C,11 and 8 peaks were obtained,respectively,which were defined as high temperature resistant and ultrahigh temperature resistant polypeptide peaks.Furthermore,the sample treated at 100°C showed strong hydrophobicity.4.Cell level study found that:high temperature resistant antioxidant polypeptide have protective effects on HUVE cells induced by H₂O₂ injury.Pretreatment with polypeptide for24 hours before injury treatment,the cell survival rate was increased by 36.2%and the LDH release was decreased by 51.37%,compared with the injury model group.And the SOD activity,CAT activity and GSH content in the cell oxidoreductase system can be effectively improve?P<0.05?.Western blot analysis showed that the polypeptides could decrease the expression of apoptotic factor Caspase-3?P<0.05?.5.Using dextran gel chromatography and DEAE ion exchange chromatography to separation and purification the high temperature resistant antioxidant polypeptide.A total of 8separated peaks were obtained.The antioxidant capacity test found that the corresponding component of peak 7 had higher antioxidant activity.And SDS-PAGE electrophoresis showed that the component contained four molecular weight polypeptides.6.High temperature resistant antioxidant polypeptide and high antioxidant active components separated by DEAE ion exchange chromatography were subjected to mass spectrometry.The results showed that according to the GO annotation,three antioxidant-related peptides were identified,namely lactoperoxidase and two?_S1-casein.The physicochemical properties,amino acid composition,hydrophobicity,signal peptide,and secondary structure of the polypeptide were predicted.The results showed that the three high temperature antioxidant peptides had higher levels of glutamic acid,leucine,valine,alanine,lysine and serine,while the content of tryptophan,methionine,histidine and cysteine were small.The three polypeptides were hydrophilic proteins composed mainly of amino acids constituting?-helices and random coils.Among them,two?_S1-casein had signal peptide.