Mechanism Of Tenderness In Yak (Bos Grunniens) Rumen Smooth Muscle During Refrigerated Storage Based On Proteomics

Yak not only provide meat(skeletal muscle)for human consumption,but also produces a mounts of by-products(stomach,intestines and other internal organs).For a long time,those by-products were mainly consumed as dishes,and has not been treated as meat.At present,the mechanism of skeletal muscle tenderness has been well researched;while smooth muscle only researched processing technology and quality changes,and there has rarely research in the mechanism of tenderness in smooth muscle.Which has become the key factor restricted the development and intensive processing of smooth muscle resources.Thus,in this paper yak rumen smooth muscle shear force,texture,pH value,drip loss,color,and cooking loss were measured to explore the quality change during refrigerated storage.The factors which affect smooth muscle tenderness development were analyzed from the perspectives of collagen and muscle fiber changes and protein degradation.Label-free proteomics method was applied to explore the effects of differentially expressed proteins on the tenderness of yak rumen smooth muscle during refrigerated storage,and bioinformatics analysis were used to clarify the mechanism of tenderness in yak rumen smooth muscle.The main findings are as follows:1.The meat quality change of yak rumen smooth muscle were determined.The results showed that,the yak rumen smooth muscle drip loss,b* value,cooking loss,gumminess were significantly increased with the extension of storage time(P<0.05);L* value,a* value,shear force,hardness and chewiness were significantly decreased with the extension of storage time(P<0.05);pH values decreased at first then increased;elastic increased at first then decreased;there was no significant difference in cohesiveness(P>0.05).The decreased rate of shear force,hardness and chewiness of yak rumen smooth muscle were 45.43%,42.71% and 30.90%,respectively.The results indicated that the tenderness of yak smooth muscle was improved during refrigerated storage.2.The main factors influencing the changes of yak rumen muscle tenderness were identified.The results showed that the yak rumen smooth muscle shear force,total collagen content,soluble collagen content,and collagen solubility were significantly decreased with the extension of storage time(P<0.05),and the decreased rate were 45.42%,20.20%,42.73%,28.12%,respectively;while MFI were significantly increased with the extension of storage time(P<0.05),and the increased rate was 170.22%;insoluble collagen content have no significantly difference(P>0.05).The correlation coefficient between shear force and MFI,total collagen content,soluble collagen content were above 0.85.Microstructure changes indicated the fiber fragmentation and collagen degradation of yak rumen smooth muscle increased during refrigerated storage.Therefore,the smooth muscle fiber degradation and weakening of connective tissue were the main reasons for smooth muscle tenderness development.3.The degradation of yak rumen smooth muscle protein during refrigerated storage were studied.The total protein content of yak rumen smooth muscle increased with the extension of storage time,and the decreased rate is 47.40%,while the content of water-soluble,low-salt and high-salt-soluble protein were increased with the extension of storage time,and the increased rate is 182.55%,39.68%,21.93%,respectively.The molecular weights of different soluble proteins of yak rumen smooth muscle were mainly distributed in 15KDa~250KDa.There have little change of the myosin heavy chain and actin of yak rumen smooth muscle,while the tropomyosin were degraded significant.The myogen of yak rumen smooth muscle increased at first then decreased.The general trend of myofibrillar is that the higher molecular weight protein bands weakening and the low molecular weight protein bands strengthening.The results showed that the smooth muscle fiber degraded during refrigerated storage.4.The differential proteome of yak rumen smooth muscle during refrigerated storage was clarified,and bioinformatics analysis was performed.There are 10947 peptides and 1353 proteins of yak rumen smooth muscle were detected by using label-free MS.A total of 212 differentially expressed proteins were identified on day 3 vs.0,day 7 vs.0 and day 7 vs.3 as determined using P<0.05 and a quantitative ratio of > 1.5 or < 0.667.Among those proteins,there were 75 actin,16 myosin,3 tropomyosin,1 myosin and 8 collagen were founded,which indicated that the main reasons of smooth muscle tenderness development is degradation of muscle fiber and the weakening of connective tissue.Bioinformatics analysis showed that those different expressed protein were mainly involved in muscle contraction,cytoskeletal regulation,apoptosis and adhesion plaque,glycolysis,which related to muscle tenderness development.5.The important proteins related to yak rumen muscle tenderness were confirmed,and the candidates indicators of yak rumen smooth muscle were selected.A total of 28 proteins were correlated with the WBSF and pH values.These proteins can be categorized into the following four groups according to their function: metabolic enzymes,muscular structural proteins,metal ion binding proteins and other proteins.Bioinformatics analysis showed those key protein were involved in focal adhesion,cardiac muscle contraction,necroptosis,and vascular smooth muscle contraction,these biological processes may influence the tenderness of yak rumen smooth muscle.The PPIs results further indicated that Calpastatin,SLC25A4,ZYX,LMOD1,TPM3,THBS4 and UQCRC1 may be candidates for indicators of yak rumen smooth muscle tenderness during refrigerated storage.6.Three different ways influencing the tenderness of yak rumen smooth muscle were explained.The first way is as follows: The anaerobic respiration of yak rumen smooth muscle during post-mortem caused the changes of pH and ion concentration,which induces the apoptosis pathway,which affect the tenderness development by the apoptotic enzymes degrade smooth muscle fiber and regulate the apoptosis process.The second way is as follows: The accumulation of phosphoric acid of yak rumen smooth muscle during post-mortem,which promoted protein phosphorylation,and regulate the development of smooth muscle tenderness by affect the activity of endogenous enzymes.The third way is as follows: The calpastatin of yak rumen smooth muscle inhibits the degradation of smooth muscle fiber by calpain,and disputed the development of smooth muscle tenderness.In conclusion,the degradation of muscle fiber and the weaken of collagen were confirmed the main reasons which affect the yak rumen smooth muscle tenderness development by research the collagen characteristics,muscle fiber degradation and muscle tissue structure change.The mechanism of tenderness in yak rumen smooth muscle during refrigerated storage were studied by proteomics,and the 28 key proteins which influence the smooth muscle tenderness were founded by correlation analysis between shear force and the differentially abundant protein,the PPIs results further indicated that calpastatin,SLC25A4,ZYX,LMOD1,TPM3,THBS4 and UQCRC1 may be candidates for indicators of yak rumen smooth muscle tenderness during refrigerated storage.Apoptosis and protein phosphorylation were the main pathways for the smooth muscle tenderness development;while calpain may have limited contribution to smooth muscle tenderness development.In this paper,the mechanism of yak smooth muscle tenderness development was revealed from three aspects: the change of smooth muscle tenderness,the change of muscle structural and protein molecular functions.The present results could provide theoretical basis and important data support for the development and intensive processing of smooth muscle.