| Tenderness is an important indicator for evaluation of beef quality. Only by taking beeftenderness improvement as a core task to promote the development of beef industry can wechange the passive situation in which the high-quality beef is dependent on import. Thetenderness of beef is affected not only by physiological impact of cattle age and species, butalso the way of treatment after slaughter. The process of which electrical stimulation effectsprotein changes in postmortem aging is still a hot debate in the meat science. But it is certainthat electrical stimulation does improve the rate of protein degradation in post-mortem muscle.The mechanism of electrical stimulation improving tenderness is complicated and needfurther research. In recent years, proteomics has become a popular way to study proteins,especially differential proteomics. By seeking, screening and identifying marker proteinsrelated to meat quality traits, differential proteomics comprehensive and in-depth insights intothe formation mechanism of meat quality beef. This study provides a scientific basis forfurther explanation of meat quality differences. Therefore, there is a very importantsignificance with proteomics method to assess meat quality.So, in this study, in addition to the determination of beef quality indicators, research ondifferential proteomics was mainly carried out. First, the system of two-dimensionalelectrophoresis gels on M. Longissimus was established, then, the differential proteins wereidentified to analysis the relations to tenderness in this system.The main results are as follows:1. Electrical stimulation influences meat quality indexs of bullsTwenty Chinese yellow crossbred bulls were selected and dividied into two groups inwhich one group was treated with electrical stimulation and slaughtered according to standardprocess. After electrical stimulation early in postmortem in the conditioning room, thecarcasses were transferred to the cutting room, and M. Longissimus were excised from thecarcasses. The indexs of temperature, pH (2h、6h、24h、72h) and shear force (1d,3d) were determined.The results showed that early in postmortem, electrical stimulation raised thetemperature, accelerated the decreasing rate of pH, reduced shear force and improvedtenderness.2. The establishment of proteomic method in M. longissimusThe methods of total protein extraction of Chinese Yellow crossbred bulls wereoptimized including lysis, loading quantity, pH range of IPG strips, protein quantification kit,dyeing method, extracting method of total protein, focusing conditions to establish theoptimum muscle protein extraction method and proteomic method in M. longissimus.The results showed that using60mmol/L as concentration of DTT in lysates and directlysis method, protein extraction is highly efficient. Due to get the wider range of proteins, theloading sample was400μg. The procedures of isoelectric focusing were24cm, pH3-10non-linear strip, CBB staining method and50v12h,100v2h,200v lh,500v30min,1000v20min,3000v20min,5000v15000vhr.3. Proteomic analysis in M. Longissimus as a result of electrical stimulationDifferential proteins were identified with PDQuest software by analyzing two-dimensional electrophoresis map to find degradated proteins markedly, including gel mapcomparison, repeatability and resolution of two-dimensional electrophoresis, differential spotsgel comparison and3D map comparison. Then the differential protein spots were identifiedand analyzed by mass spectrometry, Real-time PCR and Western blot.The results showed that twelve protein spots were down-regulated significantly at1d and3d postmortem as a result of electrical stimulation. These proteins were identified as ninetypes: Desmin, Troponin T alpha isoform, Myosin binding protein H, Creatine kinase (two),Triosephosphate isomerase (two), Peroxiredoxin-6(two), Phosphatidylethanolamine-bindingprotein, Histone H3.3-like isoform2, Methyltransferase.4. The mechanism of differential proteins as a result of electrical stimulation on tendernessThe analysis showed that proteins of nine types affect tenderness as a result of electricalstimulation through four pathways.(1) The glycolytic metabolic pathway affecting tenderness as a result of electrical stimulationMetabolic enzymes (Creatine kinase and triosephosphate isomerase) were down- regulated as a result of electrical stimulation. This indicated that electrical stimulationaccelerated the glycolytic metabolic pathway, Creatine kinase and triosephosphate isomerasewere consumed rapidly, pH value declined rapidly and finally improved tenderness.(2) The Calpains pathway affecting tenderness as a result of electrical stimulationCytoskeletal proteins (Desmin, Troponin T alpha isoform, myosin binding protein H) weredown-regulated as a result of electrical stimulation. This indicated that electrical stimulationaccelerated the Calpains pathway, Desmin, Troponin T alpha isoform, myosin binding proteinH were degradated rapidly, shear force declined rapidly and finally improved tenderness.(3) The lysosomal pathway affecting tenderness as a result of electrical stimulationPeroxiredoxin-6was down-regulated as a result of electrical stimulation. This indicatedthat electrical stimulation declined pH value rapidly until about5.4, raised sarcoplasmic Ca2+concentration and peroxiredoxin-6played a Ca2+-dependent phospholipase A2activity,resulted in the rupture of the lysosomal membrane, the release of hydrolytic enzymes, thehydrolysis of muscle proteins, finally improved tenderness.(4) The oxidative stress affecting tenderness as a result of electrical stimulationThe oxidative stress pathways of tenderization as a result of electrical stimulation mainlycomprises MAPK cascade pathway and PI3K/Akt signal pathway.1) The mechanism of peroxiredoxin-6in p38MAPK cascade pathway as a result of electricalstimulationPeroxiredoxin-6plays a peroxide oxidoreductase activity when pH is about neutral,which stimulates the proliferation and inhibition of apoptosis through p38MAPK cascadepathway. Peroxiredoxin-6was down-regulated as a result of electrical stimulation and thisindicates that electrical stimulation declined pH value rapidly until about5.4, raisedsarcoplasmic Ca2+concentration and peroxiredoxin-6played a Ca2+-dependent phospholipaseA2activity, resulted in the hydrolysis of muscle proteins and finally improved tenderness.So,electrical stimulation inhibited p38MAPK cascade pathway at some extent, inhibitedproliferation, accelerated oxidation and apoptosis and finally accelerated meat tenderization.2) The mechanism of PEBP in Ras-MAPK cascade pathway as a result of electricalstimulationPEBP was down-regulated as a result of electrical stimulation. Reactive oxygen species produced in the carcass after slaughter and activated Ras-MAPK cascade pathway. PEBPintegrated Raf-1and inhibited Ras-MAPK cascade pathway. This indicated that electricalstimulation accelerated PEBP inhibiting Ras-MAPK cascade pathway, inhibited proliferation,accelerated oxidation and apoptosis and finally improved tenderness.3) The mechanism of MT in PI3K/Akt signal pathway as a result of electrical stimulationPI3K/Akt signal transduction pathway induces proliferation, avoids apoptosis andactivates methyltransferase (MT). However, the lower expression of MT is, the lowermethylation is, the faster apoptosis is. Histone and methyltransferase were down-regulated asa result of electrical stimulation and this indicated that the expression of MT declined, thelevel of histone methylation was declined and accelerated apoptosis. So, electrical stimulationinhibited PI3K/Akt signaling pathway at some extent, inhibited proliferation, acceleratedoxidation and apoptosis and finally accelerated meat tenderization.The oxidative stress pathways of electrical stimulation indicate that meat tenderness isclosely related to oxidation and apoptosis, electrical stimulation inhibites proliferation,accelerates oxidation and apoptosis and finally accelerates meat tenderization. Specificmechanisms need to be further verified by experiments. |
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