| Tenderness is not only an important quality of meat,but also one of the factors affecting consumption.Postmortem aging process is an important way to improve the meat tenderness.The limited degradation of myofibrillar protein by endogenous enzymes such as calcium activating enzyme and apoptosis enzyme is the main reason to improve tenderness during postmortem aging.In recent years,it has been found that heat shock proteins(HSPs)can improve the tenderness of during postmortem aging.But there were few related studies on the while the research on the effect and mechanism of HSPs on tenderness during postmortem aging.Ola Tibetan sheep is one of the excellent representative breeds in the alpine pastoral areas of China.It has a large amount of movement in camping,grazing,mountain climbing and water wading all the year round,so its meat tenderness is worse than that in the breeding conditions,which affects the quality of food processing.In this paper,24 Ola Tibetan sheep were selected as the experimental objects,longissimus dorsi were selected as the test samples to study the changes of HSP70 and HSP90 expression in muscle and their relationship with meat quality.The effects of HSP70 and HSP90 on p H,ATP content,energy metabolizing enzyme and endogenous antioxidant enzyme system were analyzed from the perspective of changes of intracellular environmental factors.And the effects of HSP70 and HSP90 on apoptosis enzymes,apoptosis factors and cell morphology were clarified.By using Western blotting and immunohistochemical techniques,the intrinsic nature of HSP70 and HSP90 on myofibrillar protein degradation was revealed in vitro and in vivo.Finally,the mechanism of HSP70 and HSP90 on the tenderness of Tibetan sheep meat was clarified,which provided theoretical basis for improving its tenderness and developing and utilizing the resources of Ola Tibetan sheep.The main findings are as follows:1.The changes of HSP70 and HSP90 expression in Ola Tibetan sheep were studied during postmortem aging.The expression of HSP70 increased gradually from 0 to 12 hours after slaughter,and decreased significantly after reaching the highest value(p<0.05).The change trend of Hsp90 expression was consistent with that of HSP70.Correlation analysis showed that the expression of HSP70 and MFI was extremely significantly negatively correlated(p<0.01),the expression of HSP70 and L*value was negatively correlated(p<0.05),the expression of HSP70 and shear force,glycogen were extremely significantly positively correlated(p<0.01),the expression of HSP70 and holding water rate was positively correlated(p<0.05).The expression of HSP90 and MFI,L*were significantly negatively correlated(p<0.05),the expression of HSP90 and shear force,glycogen,holding water rate were significantly positively correlated(p<0.05).There was no significant correlation between HSP70,HSP 90 and a*,b*values.The results showed that the changes of HSP70 and HSP90had an effect on the quality of Ola Tibetan sheep meat during postmortem aging.2.It was clarified that HSP70 and HSP90 can delay the occurrence of apoptosis by affecting the intracellular environment,the activity of energy metabolizing enzymes and endogenous antioxidant enzymes.The p H value and ATP content of HSP70 and HSP90inhibitor treatment group were significantly lower than that of the control group(p<0.05).The activity of Na+-K+-ATPase and Ca2+-ATPase of HSP70 and HSP90 inhibitor treatment group were significantly higher than those of the control group(p<0.05).The SOD,CAT and GSH-Px activities of HSP70 and HSP90 inhibitor treatment group were significantly lower than those of the control group(p<0.05).The ROS level of HSP70 and HSP90 inhibitor treatment group was significantly higher than that of the control group(p<0.05)and MDA content was significantly higher than that of the control group(p<0.05).This indicated that HSP70 and HSP90 could change the p H value and ATP content of the body,reduce the activities of Na+-K+-ATPase and Ca2+-ATPase,increase the activities of SOD,CAT and GSH-Px,inhibit the release of ROS and MDA,and then it can inhibit the development of apoptosis to a certain extent.3.The effects of HSP70 and HSP90 on the morphological and ultrastructural changes of the muscle cells were studied.Compared with the control group,HSP70 and HSP90 inhibitor treatment group accelerated the speed of muscle fiber shrinkage and degradation of muscle fibers.The area and diameter of muscle fibers in the treatment group were significantly lower than those in the control group(p<0.05),and the intercellular space was significantly higher than that in the control group(p<0.05).The correlation analysis showed that the expression of HSP70 and HSP90 was related to the area and diameter of muscle fibers.There was a significant positive correlation between the diameter(p<0.01),and a significant negative correlation between the expression of HSP70 and the intercellular space(p<0.05).The results showed that HSP70 and HSP90 can effectively delay the rate of muscle fiber shrinkage,protect the degradation of myofibrillar protein,and delay the ripening and tenderizing process.4.The effects of HSP70 and HSP90 on the activity of apoptotic enzymes,apoptotic factors and apoptotic rate of muscle cells were studied.The activity of Caspase-3 and Caspase-9 in HSP70 inhibitor treatment group was significantly higher than that in the control group(p<0.05),so was the content of Bax.The level of Bcl-2 was significantly lower than the control group(p<0.05),and the ratio of Bax/Bcl-2 was significantly higher than the control group(p<0.05).The apoptosis rate was significantly higher than the control group(p<0.05).HSP90 inhibitor treatment increased Caspase-3 and Caspase-9 activity,but not significantly(p>0.05).Bax/Bcl-2 ratio was significantly higher than the control group(p<0.05),apoptosis rate was significantly higher than the control group(p<0.05).HSP70 and HSP90 can inhibit the activity of Caspases,and reduce the ratio of Bax/Bcl-2 and apoptotic rate.5.It was clarified that HSP70 and HSP90 can delay the degradation of myofibrillar skeleton protein during postmortem aging.The results of Western blotting and Immunohistochemistry showed that the expression of Desmin and Troponin-T in Tibetan sheep meat decreased gradually during postmortem aging.The expression of Desmin and Troponin-T in HSP70 inhibitor treatment group was significantly lower than the control group(p<0.05).The expression of Desmin in Hsp90 inhibitor group was lower than the control group,only at 12h and 72h was significantly different from that the control group(p<0.05),and the expression of Troponin-T in the control group was significantly different at 24h and 168h(p<0.05).The expression of Titin and Nebulin in the slaughtered Tibetan sheep meat decreased and the degradation rate increased gradually during postmortem aging.The degradation rate of HSP70 inhibitor treatment group increased by 53.68%and 47.12%respectively at 12?168h during postmortem aging,that of HSP 90 inhibitor treatment group increased by 33.07%and 32.90%respectively.The degradation rate of Titin and Nebulin in the two treatment groups were significantly higher than the control group(p<0.01).This indicated that HSP 70and HSP90 inhibited the degradation of Desmin,Troponin-T,Titin and Nebulin,and delayed the process of muscle tenderization.6.The essential reason that HSP70 and HSP90 can inhibit Desmin and Troponin-T degradation was determined.It is that HSP70 and HSP90 can inhibit the activity of Caspase-3 and?-calpain.In vitro experiments showed that Caspase-3 can degrade Troponin-T and Desmin,but the addition of exogenous HSP70 and HSP90 can obviously inhibit the degradation.The degradation of Desmin and Troponin-T was accelerated by?-calpain,and the degradation of protein was inhibited by the addition of exogenous HSP70 and HSP90 in vitro.However,HSP70 and HSP90 had no significant effect on the degradation of myofibrillar protein.It indicated that HSP70 and HSP90 could inhibit the degradation of Desmin and Troponin-T in vitro by inhibiting Caspases and Calpains.In a word,the expression of HSP70 and HSP90 has an impact on meat tenderness.HSP70and HSP90 can delay the acidification of intracellular environment,reduce the loss of ATP,improve the activity of SOD,CAT,GSH-Px.They also reduce the release of ROS,the production of MDA,the activity of Caspase-3/9 and the ratio of Bax/Bcl-2.Then they inhibit the occurrence of apoptosis.This have not good for tenderization of meat.At the same time,HSP70 and HSP90 have protective effects on the integrity of cell structure after slaughter.They can obviously inhibit the degradation of cytoskeleton protein by inhibiting Caspases and Calpains,which can delay the maturation and tenderization of meat.In this paper,the mechanism of HSP70 and HSP90 on the tenderness of Tibetan sheep meat were revealed from three aspects:cell environment,apoptosis and myofibrillar protein degradation,which provided important theoretical basis and data support for improving the tenderness and intensive processing of Ola Tibetan sheep meat. |
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