| Tenderness has been considered as one of the most important quality characteristics of fresh beef.Previous studies have demonstrated that tenderness of fresh beef are closedly relatated to the level of protein oxidation during postmortem aging.However,the mechanism about how protein oxidation regulates the tenderness of fresh beef during postmortem aging was largely unknown,particularly the mechanim of effects of different packaging methods on the tenderness of fresh beef postmortem via protein oxidation.Therefore,studying mechanism of effects of protein oxidation under different packaging methods on the tenderness of fresh beef was of practical significance.The dissertation was designed to investigate the potential mechanism for effects of different packaging methods on the tenderness of fresh beef postmortem via protein oxidation.The fresh beef was oxidized with hydroxyl free radical system(0.01 mmol·L-1 FeCl3/0.1 mmol·L-1 ascorbate/different concentrations of H2O2)to investigate the effects of oxidation on physicochemical properties,the aggragation of key cytoskeletal protein and effects of the formation of disulfide bond on the biological functions of proteins.Meanwhile,beef myosin was oxidized with different levels of hydroxyl free radicals in vitro to investigate the changes of the secondary structure of oxidized myosin and the mechanism of the degradation of oxidized myosin via ?-calpain and caspase-3.Besides,the sites of oxidation of myosin were identified.The detailed contents and results are shown as follows.1.Effects of different packaging methods on beef quality during postmortem agingEffect of air packaging and modified atmosphere packaging on the quality of fresh beef during postmortem aging was studied using vacuum packaging as control.Four left side of longissimus dorsi muscles of Chinese Northeast female yellow cattle were precooled at 4 ? for 24 h.After 24 h postmortem,beef samples(2.54 cm thick)were trimmed and mixed,and randomly divided into three groups.The beef samples were stored for 4,7 and 10 d in dark refrigeration house at 4 ? to mimic commercial storage conditions-At the end of each point,the pH values,meat color,tenderness,purge loss,centrifuge loss,cooking loss and the state of moisture distribution were determined immediately to evaluate the effect of different packaging methods on beef quality during postmortem aging.The results showed that the cooking loss of samples from air packaging and modified atmosphere packaging was not significantly different compared to vacuum packaging(P>0.05),while the purge loss of samples from air packaging and modified atmosphere packaging was significantly different(P<0.05).Air packaging presented higher pH values,while modified atmosphere packaging showed lower pH values(P<0.05).Air packaging and modified atmosphere packaging presented better improvement on L*values,a*values,b*values and higher population of free water and lower tenderness compared to vacuum packaging(P<0.05).2.Mechanism of effects of different packaging methods on beef tenderness via protein oxidation during postmortem agingThe aim of this study was to determine mechanism of effects of different packaging methods on beef tenderness via protein oxidation during postmortem aging using vacuum packaging as control.The carbonyl content and distribution,free sulfhydryl content,total sulfhydryl content,?-calpain activity,the degradation of troponin-T,desmin and titin were determined.The results showed that air packaging and modified atmosphere packaging showed greater carbonyl content,lower free sulfhydryl content,while air packaging and modified atmosphere packaging meat samples presented more fluorescence signal(P<0.05).The fluorescence signal increased in the peripheral area of cell membrane and spread to the internal cellular environment during postmortem storage compared to beef samples from vacuum packaging.The autolysis of ?-calpain decreased by the treatment of air packaging and modified atmosphere packaging,indicating a lower activity of ?-calpain.Compared to beef samples from vacuum packaging,significant lower degradation of troponin-T,desmin and titin were observed after stored in air packaging and modified atmosphere packaging(P<0.05).These results demonstrated that air packaging and modified atmosphere packaging could lead to decreased ?-calpain activity via protein oxidation,which inhibited the proteolysis of key myofibrillar proteins and the improvement of beef tenderness.3.Effects of oxidation in vitro physicochemical properties of myofibrillar proteins from beef musclesThe main objective of the study was to investigate effects of oxidation in vitro on biochemical properties of myofibrillar protein isolates(MPI)from beef muscles.MPI was incubated with hydroxyl free radical-generating systems consisting of 0.01 mmol·L-1 FeC13,0.1 mmol L-1 ascorbic acid and 0,0.2,1,5,10,20 mmol·L-1 H2O2 at 4 ? for 24 h.Protein oxidation,the surface hydrophobicity,the particle diameter and the aggregations of myofibrillar proteins were subsequently measured respectively.The results showed that hydroxyl radical oxidation caused drastically structural changes in bovine MPI(P<0.05).The carbonyl content,the surface hydrophobicity and the particle diameter of MPI significantly increased,while the free sulfhydryl group content significantly decreased with increasing hydrogen peroxide concentrations(P<0.05).The SDS-PAGE and Western blot results showed that protein oxidation caused the aggregations through cross-linking between proteins and amino acids.Proteomics study identified protein sites in which were easy to be oxidized,and oxidized catalytic activities and binding sites of enzymes which were oxidized easily were identified.Myosin,actin,titin,calpain,caspase and heat shock protein were highly sensitive to oxidation,so they easily oxidized to form disulfide bond.4.Effects of oxidation in vitro on the degradation and oxidation sites of myosin from beef musclesThe main objective of the study was to investigate effects of oxidation in vitro on the degradation of myosin from beef muscles via ?-calpain and capase-3.Myosin was incubated with hydroxyl free radical-generating systems consisting of 0.01 mol·L-1 FeCl3,0.1 mol·L-1 ascorbic acid and 0,25,50,100 ?mol·L-1 H2O2 at 37 ? for 20 min,and then?-calpain and caspase-3 were incubated at 37 ? for 30 min.The level of protein oxidation was determined by measuring carbonyl content and protein secondary structure,and the degradation of myosin was determined by SDS-PAGE and Western blot.Meanwhile,oxidation sites were identified by proteomic method.The results showed that protein oxidation systems in vitro resulted in different levels of protein oxidation leading to the significant changes of secondary structure of myosin(P<0.05).The SDS-PAGE and Western blot results showed that oxidation in vitro enhanced myosin degradation via?-calpain and caspase-3.The oxidation sites of myosin were further identified by proteomic method.Twenty two amino acids were modified by oxidation for the control group,and ten,eleven and twenty amino acids were respectively modified by oxidation for low oxidation group,medium oxidation group and high oxidation group. |
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