Mechanism Of Water-holding Capacity In Yak Longissimus Dorsi During Postmortem Aging Based On Proteomics

Yak meat is rich in nutrients,but has low water holding capacity(WHC),which affects its edible and processing quality.Therefore,control of postmortem juice loss and reduction of economic loss are crucial issues needing urgent resolution in the yak meat processing industry.Existing research suggests that changes in proteins affect the WHC of postmortem muscle,but relevant mechanism is still not entirely clear because of the complexity of protein composition.Thus,it is very important to explore the variation mechanism of postmortem WHC of yak meat by combining multiple analytical and testing methods.In this paper,the variation regularities of drip loss and cooking loss during postmortem aging were investigated by proteomic techniques using the longissimus dors i(LD)muscle of yak as the study object versus beef cattle control.The associations of metabolic enzymes,structural proteins and stress proteins with the WHC in muscle were clarified.Based on these theoretical bases,the metabolic pathways via which key proteins of yak influence WHC were revealed by bioinformatics analysis,in order to clarify the ways of and reasons for the formation of WHC in yak meat.The main findings are as follows:1.The quality indices of various WHC groups were determined,and the correlation analysis was performed.A total of 32 yaks were subjected to cluster analysis with drip loss and cooking loss as variables.The 32 samples were divided into the high water holding capacity group(HWHC)(n = 12,1.91% ? drip loss ?2.59%,29.91% ? cooking loss ?32.80%)and the low water holding capacity group(LWHC)(n = 12,3.73% ? drip loss ?4.01%,34.55% ? cooking loss ?39.72%)according to Euclidean distance 5.The samples of high water holding group were compared with those of low water group.The pH,a* value,elasticity,chewiness,total protein solubility,sarcoplasmic protein solubility and sarcomere length were higher in HWHC than in LWHC group.As a complex organism,these parameters had strong correlations.The decrease of pH caused the deformation of protein,and some of them are structural proteins and color proteins.The most important myoglobin in the protein was oxidized to high iron myoglobin,resulting in a decrease in a* value,an increase of b* value.Thus,reduced WHC of yak meat might be attributed to the decrease of pH value,protein denaturation and change of myofibrillar structure.2.The optimal conditions for two-dimensional electrophoresis(2DE)of yak LD muscle were determined.After comparison of multiple conditions,the 2DE conditions suitable for the LD muscle tissues of yak meat were: whole protein muscle extraction by lysate II method,and staining by progressive rapid step-up procedure and modified coomassie staining.Under these optimal conditions,the matching rate of protein spots was high,and high resolution images with clear background could be obtained.3.To study differentially expressed proteins on drip loss during postmortem aging of LD muscle,samples were classified according to drip loss into high drip loss(HDrip)and low drip loss(LDrip)groups.55 proteins were differentially abundant at day 0,1 and 7 during postmortem aging and identified.The identified proteins can be divided into four main functional categories: metabolic enzymes,cell structural proteins,stress related proteins and transport proteins.Among identified proteins,the volume of 24,30 and 20 spots of LDrip group were bigger than the counterpart of HDrip group,and the volume of 7,11 and 11 spots of LDrip group were smaller than the counterpart of HDrip group at days 0,1 and 7,respectively.Myosin light chain,heat shock protein 27 and triosephosphate isomerase showed a major difference between the two groups and may have the potential to be biological markers for drip loss prediction.All of three proteins decreased in intensity during storage,and they showed similar trend with proteomic results.As for GO term enrichment of cellular components,proteins related to cell,organelle and extracellular region were significantly enriched.Similarly,as for GO term enrichment of molecular function,proteins related to binding,catalytic activity and structural molecule activity were significantly enriched.Proteins involved in biological processes,such as single-organism process,cellular process and metabolic process,were found to be significantly enriched.Kyoto encyclopedia of genes and genomes pathway enrichment analysis showed 55 differentially expressed proteins were significantly enriched in the pathways,which also widely participate in multiple biological processes including carbon metabolism,glycolysis,biosynthesis of amino acids and pyruvate metabolism.Some proteins acted as core proteins and showed by gene name in biological interaction networks(BIN),such as TPI1(TIM),PKM(pyruvate kinase),LDHA(L-lactate dehydrogenase A chain),ENO3(?-enolase),NDUFB1(NADH dehydrogenase),ATP5A1(ATP synthase),HSPB1(heat shock protein),MYL(myosin light chain),TNNI1(troponin I)and DES(desmin).In protein-protein interaction analyses,one cluster showed the enzymes related to glycolytic pathway and energy metabolism,and the other one was proteins related to cell structure.4.Searching for potential predictors of cooking loss in meat is a challenging task for the meat industry.Based on the data obtained from cooking loss,LD of yak can be classified into high cooking loss(HCL)and low cooking loss(LCL)groups.26 proteins were found to be differentially abundant between HCL and LCL groups.Results showed that differentially expressed proteins on cooking loss can be attributed to metabolic enzymes,structural proteins,stress related proteins and transport protein.Among identified proteins,the volume of 14 spots of LCL group was smaller than the counterpart of HCL group,and the volume of 12 spots of LCL group were bigger than the counterpart of HCL group.The expression level of L-lactate dehydrogenase,troponin T and desmin were increased greatly significantly in HCL group.Then we used western blot and hydrophobicity analysis to validate the representative proteins.As for GO term enrichment of cellular components,proteins related to cell,organelle and membrane were significantly enriched.Similarly,as for GO term enrichment of molecular function,proteins related to binding,catalytic activity and structural molecule activity were significantly enriched.Proteins involved in biological processes,such as single-organism process,cellular process and metabolic process,were found to be significantly enriched.In protein-protein interaction analyses,one cluster showed the enzymes related to glycolytic pathway and energy metabolism,and the other one was proteins related to cell structure.In agreement with proteomic analysis,L-lactate dehydrogenase,troponin T and desmin showed as core proteins in the network.5.The differential proteome between yak and cattle muscles was clarified,and bioinformatics analysis was performed.The differentially expressed proteins were identified by mass spectrometry,and 19 kinds of differential proteins were screened out.The results showed that the major proteins attributed to inter-species difference in muscle biological characteristics between yak and cattle were metabolic enzymes,structural proteins and stress proteins.The metabolic pathways involving these proteins mainly included cellular energy metabolism,carbohydrate metabolism,genetic material signaling pathway and protein synthesis and degradation process.The physiological and biochemical reactions involving these metabolic pathways eventually led to WHC difference between yak and cattle.The proteins associated with cellular metabolism(38%),carbohydrate metabolism(31%)and energy metabolism(8%)accounted for about 77% of metabolic pathway category,thereby confirming the importance of protein metabolism in the meat quality formation of yak and cattle.6.Yak meat with varying drip and cooking losses as well as beef controls were analyzed using Venn diagram to obtain 12 kinds of proteins influencing the WHC of yak.12 kinds of proteins that affect the water retention of yak were obtained.They were LDH,CKM,beta enolase,adenylate kinase,pyruvate kinase,glyceraldehyde-3-phosphate dehydrogenase,dihydrolipoamide dehydrogenase,Desmin,troponin I,troponin T,MLC,and HSP27.The Protparam server in Ex PASy server was used to analyze the hydrophobicity of proteins which analyzed by western blot.The results showed that the score of LDH,TIM,TnT and HSP27 was higher,and the hydrophobic area was wider.The high hydrophobicity of amino acids can increase the repulsion between protein and water,which may be one of the reasons for the poor WHC in yak meat.Subcellular localization prediction using Wolf PSORT software.The differentially expressed proteins in the cells were divided into 8 positions.Differential proteins are mainly located in cytoplasm,nucleus and mitochondria.7.Three different ways influencing the WHC in postmortem yak meat were explained.The first way is as follows: Yaks produce altitude acclimatization by increasing the glycolysis to support and maintain the production of ATP,in order to adapt to the plateau environment.The glycolytic enzymes in the yak meat increase in intensity,causing declined pH in the postmortem yak meat and reduced electrostatic charge in the muscle,thereby decreasing the WHC in yak meat.The second way is as follows: Protein degradation and denaturation cause myofibril shrinkage to diminish the space capable of retaining hardly moving water.Meanwhile,structural proteins form hydrophobic bonds to reduce the WHC in yak muscle.The third way is as follows: Stress proteins regulate and maintain the water balance in postmortem muscle by maintaining the stability of myofibrillar proteins and preventing the degradation of microfilaments and other cytoskeletal proteins.Besides,the cytoplasmic change of HSP27 is more obvious under postmortem hypoxic/ischemic stress.In conclusion,this study used proteomics method to study on the change of protein in yak meat after slaughter.Cattle was used as control.12 key proteins that affect the water retention of yak were found.In this paper,three different ways to influence the water retention in the yak meat were discussed in different levels of protein molecules,muscle cells and WHC of muscle.To clarify the mechanism of water retention of yak meat in the course of post slaughter maturation,the purpose of this study was to provide a theoretical basis for the explanation of the differences in the biological characteristics and meat quality of yak meat.