Effect Of Structural-changes Induced By Pyrophosphate On The Cross-linking Action Of Pork Myosin By TGase

Transglutaminase?TGase?was widely used in meat products to catalyze the acyl transfer reaction between glutamine?Gln?and lysine?Lys?residues to form a highly viscous gel network structure and thereby improve the quality of meat products.The cross-linking effect of TGase is closely related to the structure of substrate protein.However,the occurrence of protein oxidation?during storage,transportation and processing?and the addition of phosphate in meat products could change the structure of muscle proteins and might change the available content of Gln and Lys residues for TGase.Therefore,protein oxidation and phosphate was likely to affect the cross-linking pattern of TGase.Myofibrillar protein and myosin were oxidatively stressed in Fenton hydroxyl radicalgenerating system?Fe C13/ascorbic acid/H₂O₂,p H 6.25?with different H₂O₂ concentrations and then subjected to TGase?E:S=1:20?.The effects of pyrophosphate-induced myosin structure changes on the cross-linking of TGase were studied.The main research contents and results were as follows:The effect of structural-changes induced by sodium pyrophosphate?TSPP?on the cross-linking degree of myosin by TGase was studied.The results showed that for myosin without TSPP treatment?–TSPP?,the blocking degree of two active sulfhydryl SH1 and SH2 in myosin head was significantly increased with the elevated H₂O₂ concentration?1⁵0mmol/L??P<0.05?.The trends of surface hydrophobicity increased first and then decreased.The content of ?-helix decreased from 48.74% to 44.51%.TSPP treatment?+TSPP?reduced the oxidative modification of SH1 and SH2,and decreased the surface hydrophobicity of myosin?P<0.05?.The content of ?-helix structure increased to 49.83% after TSPP treatment.It can be see that TSPP could protect the secondary structure of myosin during oxidation.The content of ?-helix significantly decreased when treated with TGase?P<0.05?,while surface hydrophobicity significantly increased?P<0.05?.The changes of ?-helix and surface hydrophobicity were further deepened after TSPP treatment.The relative changes of ?-helix structure caused by TGase were related to the oxidation degree.For –TSPP samples,The relative changes of ?-helix structure caused by TGase in sequence were mild oxidation?1mmol/L H₂O₂?> unoxidized>severe oxidation?30 and 50 mmol/L H₂O₂?,while for +TSPPsamples,the relative changes of ?-helix increased with H₂O₂ concentration.Free amino and SDS-PAGE results showed that TSPP can promote cross-linking reaction catalyzed by TGase.The effect of myosin structure changes induced by TSPP on the active substrate Gln residues cross-linked by TGase showed that the active sites of TGase cross-linking ware in S1?Gln-613?and LMM?Gln-1498?in –TSPP and nonoxidized sample,while S1?Gln-613?was no longer the active site of TGase cross-linking in severe oxidation?50 mmol/L H₂O₂?.For+TSPP samples,the sites were in S1?Gln-558 and Gln-613?,S2?Gln-1361 and Gln-1373?,LMM?Gln-1422/Gln-1425 and Gln-1498?were the active sites of TGase cross-linking in nonoxidized sample,but Gln-1361 was no longer the active site in severely oxidized sample.The effect of TSPP and oxidation on the secondary structure of myosin catalyzed by TGase was studied.The results showed that no matter with or without TSPP,the storage modulus?G'?and gel strength of myofibrillar protein gel gradually reduced with enhanced H₂O₂ concentration,but showed resilience after TGase treatment.The amplification of G' and gel strength exclusively induced by TGase decreased with enhanced H₂O₂ concentration for both–TSPP and +TSPP samples.Compared to –TSPP samples,the gain of G' and gel strength in+TSPP samples exclusively induced by TGase was higher?for 72.19% and 63.11%?,but the gain decreases sharper when attacked by hydroxyl radicals.It suggested that TSPP treatment made the inhibiting effect of protein oxidation on the improvement of gel properties exclusively induced by TGase stronger.Correlation between TGase cross-linking degree,protein structure and gel properties showed that for –TSPP samples,the changes in cross-linking degree and gel properties caused by oxidation or TGase were significantly correlated with the amino acid modification and secondary structure changes induced by oxidation?P<0.05?,while for +TSPP samples,the changes were no longer related to the change of secondary structure.It could be seen that the correlation between the cross-linking degree of TGase and the protein gel properties and secondary structure was weakened under the condition of TSPP.In summary,this study illuminated that the effects of pyrophosphate-induced myosin structure changes on the cross-linking degree of TGase and gelation properties of TGase mediated myofibrillar protein.The structure factors affecting TGase cross-linking and thequality of products were also revealed.This study provided a theoretical basis for the application of TGase or other cross-linking enzymes and the quality of meat products.