Mechanism Of Meat Color Stability Regulated By Protein Phosphorylation

This study investigated the effects of protein phosphorylation on meat color stability.Ovine longissimus thoracis et lumborum(LTL)muscle was used in this study.Phosphatase and protein kinase inhibitors were added to regulate the global phosphorylation of sarcoplasmic proteins,and the influence of phosphorylation on meat color stability was studied.Comparative profiling of sarcoplasmic differential phosphoproteins in ovine muscle with different color stability was investigated by using SDS-PAGE & LC-MS/MS,and the pathways by which protein phosphorylation regulates meat color stability was also analyzed.The key phosphorylated proteins and its phosphorylation site which regulates meat color stability were quantitatively analyzed by using Tandem mass tag(TMT)technology.A in vitro model was established to regulate the phosphorylation level of myoglobin,and the effects of phosphorylation on the redox stability of myoglobin was studied.The results are as follows:(1)This study suggests that protein phosphorylation play a native role in the regulation of meat color stability.Phosphatase and protein kinase inhibitors were added to minced ovine LTL muscle to regulate the phosphorylation and dephosphorylation of proteins.The data obtained show that the change of p H values and the accumulation of lactate accumulation in postmortem muscle were related to protein phosphorylation.Analysis of meat color and the relative content of myoglobin redox forms revealed that meat color stability was inversely related to the phosphorylation of sarcoplasmic proteins.(2)The possible pathways by which protein phosphorylation regulates meat color stability were investigated in the present study.The phosphorylation of sarcoplasmic proteins in postmortem muscles with different color stability are measured by SDS-PAGE & Pro-Q & Ruby.Although no difference was observed in the global phosphorylation level of sarcoplasmic proteins,difference was determined in the phosphorylation levels of individual protein bands from muscles with different color stability.LC-MS/MS identification of phosphoproteins showed that most of the color stability-related proteins were glycolytic enzymes.Interestingly,the phosphorylation level of myoglobin was inversely related to meat color stability as the phosphorylation of myoglobin increased,color stability decreased and metMyoglobin content increased.In conclusion,the present study reveals that the possible mechanisms of protein phosphorylation regulates meat color stability may include the following pathways.Fist,protein phosphorylation regulates the activities of glycolytic enzyme,which then impact the glycolysis metabolism process and thus have an influence on meat color stability.Second,the phosphorylation of myoglobin might regulate its redox stability and thus has an influence on meat color stability.(3)The key phosphoproteins and its phosphorylation site which regulates meat color stability were identified in this study.A total of 3412 phosphopeptides and 1070 phosphoproteins were identified by using Tandem mass tag(TMT)technology.Bioinformatics analysis indicated that most of thosecolor-related phosphorylation proteins were involved in carbohydrate metabolic,and the phosphorylation on 133 th serine residues of Mb play negative roles in the regulation of meat color stability.In summary,this study indicated that the phosphorylation of those selected carbohydrate metabolic enzymes and myoglobin on particular serine residues may play crucial roles in the regulation of meat color stability.(4)Protein phosphorylation involved in meat color development by regulating the redox stability of myoglobin was verified in this study.The pure metmyoglobin from skeletal muscle was used and reduced by sodium dithionite.After that,phosphatase were added to catalyze the dephosphorylation of myoglobin in vitro.The data showed that the phosphorylation level of myoglobin was lower(P < 0.05)in the experimental group than that in the control group,and the redox stability of myoglobin was inversely related to its phosphorylation.The results of circular dichroism spectrum indicated that the secondary structural stability of Mb was increased after dephosphorylation.This study indicated that the secondary structural stability was decreased and the autoxidation rate of myoglobin was increased after phosphorylation,which maybe one of the reasons by which protein phosphorylation regulates meat color stability.