Identification Of Potential Biomarkers Of DFD Beef

Dark firm dry(DFD)meat is one of the major quality defected meat associated with abnormal postmortem metabolism.Consumers tend to reject such meat as it is perceived to be from old or poorly-handled animals due to its dark color,tough texture,undesirable flavor and short shelf-life.DFD meat has been the subject of extensive research,however,its exact biochemical mechanism and biomarkers are not clear and it still occurs and causes significant financial losses globally in the fresh beef market.In order to find the mechanisms and biomarkers of this defect different techniques have been employed.The study was assessed through four experiments.First experiment investigated the basic meat quality of DFD beef and developed the association of color with other quality traits.Beef longissimus thoracis(LT)was assessed and classified into one of three groups based on the pH 24 h postmortem:normal(pH?5.70),atypical DFD(5.70<pH?6.09)and typical DFD(pH>6.09)beef.Samples were stored at 4°C for 7 days and used in all four experiments.Typical DFD beef showed the lowest glycolytic potential,L~*,a~*,b~*,chroma and oxymyoglobin accompanied with the highest water-holding capacity,hue angle and deoxymyoglobin values on day 1 of storage.Glycolytic potential was positively correlated with chroma,R630/580 and oxymyoglobin but was negatively correlated with pH and hue angle.Water-holding capacity was directly related to deoxymyoglobin levels.Atypical DFD beef showed a higher shear force value that was positively associated with metmyoglobin levels.In conclusion,DFD beef has many meat quality defects with compromised glycolytic potential and its color was not only affected by ultimate pH but also related to shear force,glycolytic potential and water-holding capacity.Further research should focus on delineating the underlying mechanisms to mitigate its occurrence.Second experiment was designed to elucidate the mechanisms underlying the formation of DFD beef using protein phosphorylation.Phosphorylation of sarcoplasmic and myofibrillar proteins was assessed on day 1,2,3,5 and 7 postmortem.Results showed that global phosphorylation of sarcoplasmic proteins was higher in normal beef on day 1 postmortem and of myofibrillar proteins was higher in atypical DFD beef on day 1 and 2 postmortem(P<0.05).The phosphorylation levels of sarcoplasmic and myofibrillar proteins of all groups were decreased with increase of postmortem time.Sarcoplasmic protein bands with different phosphorylation levels were mainly identified as glycometabolism and stress response proteins and myofibrillar protein bands revealed sarcomeric,metabolic and heat shock proteins.This study indicated that protein phosphorylation of several metabolic,heat shock and sarcomeric proteins were linked with the development of DFD beef and provided information on the diagnostic biomarkers of this defect.Third experiment explored the non-targeted metabolomics of the energy metabolites to explain the biochemical basis linked with the formation of DFD beef.Muscle samples from the normal,atypical DFD and typical DFD beef on day 1 postmortem were analysed by ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry(UHPLC Q-TOF/MS).A total of 9033 and 6951metabolites were obtained,in which 76 and 87 compounds were identified as significantly different metabolites in positive and negative ion modes respectively.The KEGG enrichment analysis showed that differential metabolites were mainly found in central carbon metabolism,purine metabolism,protein digestion absorption,glycine,serine and threonine metabolism,and aminoacyl-tRNA biosynthesis.Several significantly different metabolites involved in different biochemical responses including animal stress were linked with the meat quality of DFD beef,which can be used as potential biomarkers of this defected beef.In conclusion,this study further explained the findings of experiment 2 and it was found that,not only glycolysis,but several postmortem metabolic pathways especially central carbon metabolism have been involved in formation of DFD beef.Fourth experiment was aimed to confirm the role of heat shock proteins(HSP)identified in experiment 2,or animal stress recognized in experiment 3 in determination of postmortem metabolism and development of meat quality of DFD beef.Samples were tested for HSP 27,HSP 70 and HSP 90levels on day 1,3,5 and 7 postmortem and for meat quality parameters and glycolytic metabolites on day2 postmortem.Results showed that level of HSP 27 was higher in atypical DFD on day 1,however,HSP70 was higher in typical DFD on day 1 and 3 and HSP 90 was lower in normal beef on day 1 compared with other two groups.HSP 27 presented a positive correlation with the shear force values.HSP 70 and HSP 90 behave similarly and presented direct relationship with pH and water-holding capacity and indirect relationship with color(L~*,a~*and b~*),glycogen and lactate contents.The study showed that heat shock proteins played a major role in development of meat quality of DFD beef,which can used as biomarker of this defect.In conclusions,it was found that DFD beef has many meat quality defects with compromised glycolytic potential.Various metabolic,sarcomeric and heat shock proteins and metabolites were involved in development of processes underlying the formation of this defect.Furthermore,glycogen phosphorylase,glyceraldehyde-3-phosphate dehydrogenase,phosphoenolpyruvate,3-phospho-D-glycerate,succinate,dihydroxyacetone and alpha-D-galactose 1-phosphate,HSP 70 and HSP 90 were identified as potential biomarkers to identify DFD beef.