Effect Of Hydroxyl Radical Oxidation System On The Water Holding Capability Of Pork Myofibrillar Protein Gel

As an important part of daily animal food on people’s table,pork is an important source of people’s minerals,nutrients and fatty acids,and it has a profound impact on human health.Although China is currently a major producer and consumer of pork,there are still some disparities between our country’s meat industry and developed countries or developed regions.Myofibrillar proteins（MPs）are the main component of total muscle protein,and their quality have critical influence on the quality characteristics of processed meat products.However,MPs are vulnerable to reactive oxygen species（ROS）attack during processing,which changes the water holding capability（WHC）of meat products,thus affecting the edible quality of meat products and reducing the economic benefits of meat industry.As a kind of free radical widely existing in meat processing,hydroxyl radical（·OH）can lead to rapid oxidative modification of proteins.There have been many studies about the effects of oxidation on the properties of protein gel in recent years.However,there are few studies about the interaction between different oxidation indicators under oxidation conditions,and the in-depth influence of oxidation on the formation of pork MPs gel is still not fully understood.In addition,further explanations of what components of MPs are changed accompanied by oxidation and the influences of the changed components on MPs gel’s WHC are also needs to be provided.Herein,the pork MPs are used as the research object in this study.The effects of·OH oxidation system on the physicochemical properties of pork MPs are discussed in depth,and the reasons of changes in WHC of pork MPs gel induced by oxidation are systematically analyzed.The experimental results can provide more biochemical information for the changes of pork MPs under oxidation conditions,then clarify the ways that oxidation leads to changes in the components and structure of MPs.And theoretical reference is given for better controlling protein oxidation and improving WHC in the production of gel-type meat products（such as gel-type meat products with lean meat as the main raw material,thermal gel products with muscle protein,gel-type products with complex muscle protein）.The main conclusions are as follows:（1）The pork MPs are used as the research object,and the effects of·OH oxidation system on the physicochemical properties of pork MPs gel（focus on WHC）and MPs are investigated.The results show that the physicochemical properties of MPs are not significantly changed conducted by proper oxidation treatment{（Hydrogen peroxide,H₂O₂）<1 mmol/L}.Additionally,the gel prepared by properly oxidized MPs presents a uniform and dense microstructure,the springiness and WHC are increased to a certain extent.When the concentration of H₂O₂is 20 mmol/L,the components and structure of MPs are changed significantly.The carbonyl content,average particle size（d50）,aggregates content and surface hydrophobicity of MPs are increased significantly（p<0.05）,while the sulfhydryl content of MPs,the endogenous fluorescence intensity,the peak value of ultraviolet absorption spectrum and the absolute value of Zeta potential（MPs solution）are decreased obviously（p<0.05）.The Brunauer-Emmett-Teller（BET）specific surface area and free water percentage of the gel prepared by MPs treated with 20 mmol/L H₂O₂are increased significantly（p<0.05）,the springiness and WHC are decreased by 16.18%and 21.60%respectively.MPs can be formed a uniform and dense network gel structure conducted by proper oxidation treatment（H₂O₂<1 mmol/L）,as well as improving the WHC and springiness of MPs gel.However,the MPs treated by strong oxidation treatment（H₂O₂=10 mmol/L or 20 mmol/L）can form a large number of aggregates,and the formed MPs gel with rough microstructure,and the WHC and springiness are significantly reduced.（2）The effects of different oxidation levels on the gel-forming process of pork MPs are explored.The results reveal that with the increased concentration of H₂O₂,the content of dityrosine in MPs is gradually increased,and the results of exclusion chromatography show that the content of large molecular weight polymers in MPs is also increased.The storage modul（G’）and loss moduli（G’）values of MPs are totally presented a decreasing trend with the increased concentration of H₂O₂.From electrophoresis patterns,the cross-linking aggregation of MHC and actin in MPs as well as MPs gel are increased with the increased concentration of H₂O₂,and the protein dissolved in the gel-forming process is increased as well.Strong oxidation conditions（H₂O₂>10 mmol/L）lead to excessive disordered cross-linking and aggregation of MPs,which also increases the relative content of heat shock protein 90（Hsp90）in response to ROS attack on the protein.During the thermal gel-forming process,the strongly oxidized MPs can hardly produce good intermolecular interaction due to their over disordered cross-linking and aggregation,which causes the increased content of protein dissolution.The resulting MPs gel has poorer structural properties,lower springiness and lower WHC.（3）The effect of in vitro oxidation on aggregation of pork MPs is studied.The results indicate the structure of MPs is gradually unfold under the proper oxidation treatment（H₂O₂<1 mmol/L）.Meanwhile,α-helix is decreased,whileβ-sheet and solubility are increased to a certain extent,but the difference is not significant（p>0.05）.The chemical bond,X-ray diffraction（XRD）patterns and the content of basic amino acids of MPs are also no great change（p>0.05）after treated by proper oxidation.When the concentrations of H₂O₂are 10 and 20 mmol/L,the relatively ordered structure of MPs such asα-helix andβ-sheet conformation are declined obviously（p<0.05）,while the relatively flexible structure such asβ-turn and random coil conformation are increased markedly（p<0.05）.The contents of ionic bonds and hydrogen bonds for maintaining the stability of MPs structure are reduced remarkable（p<0.05）,nevertheless,the contents of hydrophobic interactions and disulfide bonds are increased markedly（p<0.05）.The content of basic amino acids in MPs is decreased more,and MPs’isoelectric point（p I）is showed a tendency to move to the acidic region.The percentage of large particle size of MPs is gradually increased under strong oxidation conditions,confirmed by particle size distribution and laser confocal.Strong oxidation（H₂O₂>10 mmol/L）makes the effects of cross-linking and aggregation of MPs in solution（such as changes in secondary structure,the increase of hydrophobic interactions and disulfide bonds,etc.）,which are greater than the effects that promote its stability（such as changes in p I）.Eventually,the particle size of MPs is increased and a large number of disordered cross-linked polymers are formed,and then affect its gel-forming power.（4）The effects of different oxidation levels on the components of pork MPs and the interaction between differentially abundant proteins（DAPs）are investigated by Tandem Mass Tag（TMT）-labeled quantitative proteomics technology.The results demonstrate that the structure and components of MPs are not significantly changed conducted by proper oxidation treatment（H₂O₂<1 mmol/L）,and the interaction between differential proteins is also weaker.The differences of structural proteins,transport proteins,oxidative phosphorylation and glycolysis related enzymes in MPs are significantly increased under strong oxidation conditions（H₂O₂>10 mmol/L）.And the change trend of these DAPs is consistent with the change trend of MPs gel’s WHC.Compared with the control group,the interaction of DAPs in MPs is the strongest in the strongly oxidation group,and the number of interaction network nodes is the largest.H₂O₂can induce the interaction between the enzymes in MPs and other proteins under the strong oxidation conditions,and the structure and components of MPs are visibly changed under the combined effects of H₂O₂and endogenous enzymes.Myosin,keratin,oxidative phosphorylation and glycolysis related enzymes have significant differences between proper and excessive oxidation,which can be used as predictors for oxidation degree of MPs.（5）The effects of different oxidation levels on the relative contents of endogenous enzymes which are bound by MPs and the degradation of pork MPs are studied by Western Blot.The results present that the band relative optical densities of phosphorylase b kinase,sarcoendoplasmic reticulum calcium ATPase and phospho-myosin heavy chain（MHC）9 are tended to increase with the increased concentration of H₂O₂.When H₂O₂>10 mmol/L,the band relative optical density of phosphorylase b kinase is increased slowly,but the band relative optical densities of phospho-MHC 9 and sarcoendoplasmic reticulum calcium ATPase began to show a downward trend.The band relative optical densities of MHC 14 andα-actin 1 showed a downward trend as the concentration of H₂O₂increased.The autolysis ofμ-calpain is delayed under strong oxidation conditions（H₂O₂>5 mmol/L）,and then the action time ofμ-calpain on protein is prolonged.In the in vitro oxidation system induced by H₂O₂,the oxidation degradation of MPs is affected by both·OH and endogenous enzymes,the strong oxidation treatment can eventually leads to a decrease in WHC of the gel prepared by MPs.In summary,the effects of the·OH oxidation system on the WHC of pork MPs gel are mainly as follows:ROS will not cause significant changes in the components and structure of MPs under proper oxidation treatment（H₂O₂<1 mmol/L）.The content of protein dissolution is less during the thermal gel-forming process,and MPs can form a uniform and dense network gel through the orderly arrangement of disulfide bonds between molecules.MPs gel has smaller pores,larger capillary forces,and higher immobilized water content,so its WHC is better.The oxidation modification of MPs conducted by ROS have increased obviously under the strong oxidation conditions（H₂O₂>10 mmol/L）,as a result,the components and structure of MPs are changed significant.And the differences of relative contents of endogenous enzymes bound by MPs are also increased markedly,some endogenous enzymes are also involved in the oxidation degradation of MPs under strong oxidizing conditions.The modification of the amino acid side chain of MPs is increased dramatically conducted by strong oxidation conditions,and the sulfhydryl groups are consumed in large quantities.A large number of disordered aggregates in MPs are generated by disulfide bonds,hydrophobic interactions and tyrosine cross-linking,some charges on the surface of MPs are masked,and these results lead to the solubility of MPs decreased.Due to the strong oxidation treatment,a large number of disordered aggregates are generated by MPs,and the sulfhydryl groups are consumed in large quantities.In the gel-forming process,MPs molecules can not be arranged in an orderly arrangement by producing more disulfide bonds,which leds to the content of protein dissolution is increased.The formed MPs gel has larger pores,and the free water content is increased,which eventually leads to the decreased WHC of the MPs gel.Accordingly,in the production of gel-type meat products,in order to improve the texture characteristics and WHC of products,in addition to the need to take measures to reduce the impacts of ROS on MPs components and structure,it can also regulate the cross-linking way of MPs.And then a uniform dense network structure will be generated to improve the WHC of gel-type meat products.