The Mechanism Of Calpain-mediated Dietary Antioxidant Reduce Drip Loss In Lamb Meat

In order to study the effects of different antioxidants on the mechanism of drip loss in lamb meat during storage,two exogenous antioxidants,vitamin E(VE)and lycopene(LP)were used in this study.Animal experiments were divided into two parts.The first part:forty healthy male Hu lambs were selected,which had the same genetic background,similar birth weight,and weaned at about 3 months of age.They were randomly divided into 4 groups,each of which was fed in a single pen.The VE levels were 0(CON),200(VE200),400(VE400),2000(VE2000)IU/d added to the basal diet.The second part:thirty male Hu lambs(3 months old)with identical genetic background and similar body weight and healthy weaning Hu sheep were randomly divided into 3 groups with 10 in each group.Set the LP levels to 0(CON),200(LP200),and 400(LP400)mg/kg basal diets.After three months of feeding trials,the sheep were all slaughtered.The sample of longissimus dorsi(LD)in lamb was taken for analysis of meat quality,antioxidant properties,calpain levels,and proteomics.Experiment 1:Effect of dietary antioxidants on lamb quality.A portion of the LD muscle was stored for 0,1,3,5,and 7 days at 4? to determine drip loss,pH,color,and nutrient contents.The results showed that pH showed the highest value at day 0(P<0.05),and other time periods had no significant effect on pH(P>0.05).Compared with the control group,the VE200 and VE400 groups significantly reduced the L*value and drip loss on the day 7(P<0.05),and increased the value of a*(P<0.05).The VE200 group significantly reduced drip loss on dayl(P<0.05).The value of a*showed the highest value on day 1 and the lowest value on the day 5.Addition of LP in diets significantly?increased pH,a*,b*,H*(P<0.05),decreased L*,crude ash content,and drip loss on day 7(P<0.05).The drip loss was reduced on day 1,and the crude protein content was increased significantly(P<0.05)in the LP200 group lamb meat.The b*value,H*value and drip loss gradually increased with time(P<0.001).Experiment 2:Effect of dietary antioxidants on oxidation resistance of lamb.The LD muscle sample was stored at 4? for 0,1,3,5,and 7 days to determine thiobarbituric acid-reactive substance(TBARS)values,carbonyl and thiol groups,and VA and VE content.The results showed that the interaction effect of VE and time significantly affected the content of VE and TBARS in lamb(P<0.01).VE significantly increased total antioxidant capacity,catalase,VE and thiol content in lamb(P<0.05),and decreased TBARS and protein carbonyl content significantly(P<0.001).Compared with the control group,VE200 and VE400 significantly increased the content of SOD and GSH-Px in lamb(P<0.05),and VE200 significantly increased VA content in lamb(P<0.05).The interaction between LP and time significantly affected the content of TBARS and carbonyl in lamb(P<0.01).Compared with the control group,LP could significantly increase the activity of T-AOC,CAT,SOD,GSH-Px and VE content,reduce TBARS and carbonyl content significantly(P<0.05),LP200 significantly increased VA content in lamb(P<0.001).The content of VA and VE,TBARS and carbonyl content increased significantly with time(P<0.05),and the sulfhydryl content decreased with time(P<0.001).There was a significant positive correlation between lipid oxidation and drip loss(P<0.001).Experiment 3:Regulation of dietary antioxidants on the expression of calpain gene and protein in lamb meat.The LD muscles were stored in liquid nitrogen and then frozen at-80? for gene expression determination.The samples of LD were stored at 0,1,3,5,and 7 days during storage,respectively.The results showed that compared with the control group,the VE200 and VE400 groups significantly increased the relative expression of Calpain-1(P<0.05).The LP400 group significantly increased the relative expression of Calpain-2 mRNA(P<0.05).Under the effect of VE,the level of Calpain-1 reached the highest value on the day 5(P<0.05).Under the action of VE and LP,the relative expression of Calpain-1 mRNA was negatively correlated with drip loss significantly,the expression level of Calpain-2 was decreased on the day 1(P<0.05),and remained relatively stable during the 1-7 days.Experiment 4:Isobaric Tags for Relative and Absolute Quantitation(iTRAQ)technology analyzes the effect of vitamin E on differentially expressed proteins(DEPs)in lamb meat.After the sheep were slaughtered,lambs longissimus muscle samples were collected in control group and in VE200 group at 0,5,and 7d.The results identified 7385 unique peptides and 1786 proteins.Using FoldChange>1.2 or<0.83 as the screening standard for differential protein expression,the expression levels of the three treated(CON-D0/VE-D0,CON-D5/VE-D5,CON-D7/VE-D7)proteins were compared in pairs.283 differential proteins,CON-D5 and VE-were obtained in CON-DO and VE-DO groups.A total of 228 differential proteins were obtained in the D5 group,and 245 differential proteins were obtained in the CON-D7 and VE-D7 groups.These proteins were identified to be involved mainly in ribosomal protein pathways,oxidative phosphorylation,signal transduction,and regulation of the actin cytoskeleton.The functional annotations indicated that 8 DEPs were related to the antioxidant properties and protein degradation of lamb.The antioxidant properties were mainly related to NDUFB7(NADH ubiquinone oxidoreductase subunit B7),COX5B(Cytochrome c oxidase subunit 5B),CYC1(Cytochrome c1),OGDH(Oxoglutarate dehydrogenase),and SUCLG2(Succinate--CoA ligase,GDP-forming subunit beta),and the protein degradation was mainly related to calpain-regulated TPM3(Tropomyosin 3),TNNC1(Troponin C1),and MYL2(Myosin light chain 2).The parallel reaction monitoring(PRM)results indicate the reliability and validity of the iTRAQ results.Experiment 5:iTRAQ technology analyzes the effect of lycopene on DEPs in lamb meat.After the sheep were slaughtered,the longissimus muscle samples were collected in control group and in LP200 group at 0,5,and 7 d.The test results identified more than 7385 unique peptides and 1766 proteins.Using FoldChange>1.2 or<0.83 as the screening criteria for differential protein expression,the expression levels of the three treated(CON-D0/LP-D0,CON-D5/LP-D5,CON-D7/LP-D7)proteins were compared in pairs.343 differential proteins,CON-D5 and LP-were obtained in the CON-DO and LP-D0 groups.A total of 315 differential proteins were obtained in the D5 group,and 214 differential proteins were obtained in the CON-D7 and LP-D7 groups.These differential proteins are mainly enriched in carbon metabolism,adrenergic signaling in cardiomyocytes,proteasomes,oxidative phosphorylation,citric acid cycling,pentose and glucuronide interconversion,regulation of actin cytoskeleton,retinol Metabolism and other signal pathways.Eleven different proteins were screened,which were mainly related to protein metabolism and protein turnover.Among PSMC2(Proteasome 26S subunit),PSMA3(Proteasome subunit alpha type),CACNB1(Calcium voltage-gated channel auxiliary subunit beta 1),TPM3,TNNC1,MYL2,and MYL4(Myosin light chain 4)were all associated with calpain and could be used as markers for the detection of drip loss.In summary,addition of vitamin E and lycopene in' the diet could improve antioxidant performance,reduce drip loss,and improve lamb quality during storage.Vitamin E and lycopene regulate calpain through oxidized proteins(NDUFB7,COX5B,CYC1,OGDH,SUCLG2)and metabolizing proteins(SUCLA2,SUCLG2,ALDH1A1,PSMC2,and PSMA3),respectively,which are activated by myofibrillar proteins(TPM3,TNNC1)MYL2)degrades and reduces the loss of dripping mutton.