| In order to meet safety and food quality requirements,meat is usually subjected to heat treatment before consumption.Salt is an essential ingredient in meat processing,and different processing techniques and additives can cause changes in food quality and the protein structure of meat.Traditional Chinese meat products have a variety of types,and different processing temperatures and Na Cl concentrations may be used.These can cause changes in the protein molecular structure,which can affect its biological utilization.However,the effects of different heating temperature and Na Cl concentration on the structure change and digestion of meat protein have not been systematically studied,the mechanism of the effect of heating and Na Cl on protein structure,oxidation degree and digestibility is not clear,and the relationship between the three remains to be explored.Therefore,this study investigated changes in the structure,oxidation degree,and protein digestibility of beef myofibrillar protein under different heating temperatures(40,55,70,85,100,115°C)and Na Cl concentrations(0,0.2,0.4,0.6,0.8 M).Correlations were established between the three factors to preliminarily explore the interaction between protein and the three factors during heating.This provides a theoretical basis for further research on the correlation between protein oxidation and digestibility in meat after heat treatment,promotes the processing and development of nutritious and healthy meat products,and has positive significance for the meat industry.The main findings and conclusions are as follows:1.Effect of heating temperature on the structure and oxidation degree ofmyofibrillar proteinsUsing beef myofibrillar protein as the research object,the effects of different heating temperatures on protein structural changes and oxidative degree were analyzed.The study found that heating temperature was significantly positively correlated with protein carbonyl content and dityrosine content,and negatively correlated with thiol content and solubility.Solubility was highly negatively correlated with carbonyl content,dityrosine content,and surface hydrophobicity index.Between 40-85℃,with increasing heating temperature,myofibrillar protein solubility decreased,surface hydrophobicity increased,and endogenous fluorescence intensity decreased,indicating that hydrophobic groups were exposed on the protein molecule surface at this time.The α-helical content of secondary structure increased,while the β-sheet content decreased,indicating that appropriate heating promotes the opening of protein β-sheet structure and transition to α-helical structure,demonstrating protein structure extension.However,when the heating temperature exceeded 85℃,protein solubility and surface hydrophobicity decreased,hydrophobic groups began to aggregate,the α-helical content of secondary structure transformed into β-sheet,and the increased β-sheet structure and dityrosine content made the protein easy to aggregate.Meanwhile,the significant increase in carbonyl and dityrosine content and the decrease in active thiol content indicated that heating promoted protein oxidation.SDS-PAGE results showed that with increasing heating temperature,the large molecular bands gradually became lighter or disappeared,while new small molecular bands appeared,indicating that heating promotes protein denaturation and degradation.2.Effect of heating temperature on the digestion properties of myofibrillarThe effects of different heating temperatures on the digestion of beef myofibrillar protein were investigated using an in vitro digestion simulation.The results showed that the digestion rate of myofibrillar protein gradually increased from 40-85°C,reaching the highest level of 59.54% at 85°C.The decrease in β-sheet content from40-85°C indicated that the protein structure unfolded under mild oxidation,exposing previously hidden cleavage sites on the molecular surface,making it more conducive to the specific site binding of gastric and pancreatic proteases,thereby promoting protein digestion.The solubility decreased from 85-115°C due to the formation of disulfide bonds and hydrophobic interactions that caused protein aggregation.In addition,the formation of carbonyls reacted with non-oxidized amino groups of proteins,burying the specificity recognition sites of digestive enzymes,leading to a decrease in protein digestion.During digestion,the α-helix content gradually increased,while the β-sheet content decreased.The number of β-turns first increased and then decreased,indicating that the digestion of protein by gastric and pancreatic proteases mainly destroyed its β-structure.These results indicate that appropriate heating and protein extension caused by oxidation are beneficial for digestion,while excessive heating and protein aggregation caused by oxidation are not conducive to digestion.3.Effect of Na Cl on the structure and oxidation degree of heat-induced myofibrillarA study was conducted on beef myofibrillar protein to investigate the effect of Na Cl concentration on the structure and oxidation level of heat-induced myofibrillar protein.The results showed that Na Cl can increase the solubility of the protein,and low concentrations of salt ions cause the structure to unfold,which is more pronounced at 85°C.Specifically,myofibrillar protein exhibited maximum extension in a 0.4 M salt solution after being heated to 85°C.However,as the temperature increased or the Na Cl concentration increased,both led to severe protein oxidation and aggregation.When not heated,the hydrophobicity of the surface and β-sheet in the secondary structure gradually decreased from 0-0.4 M,and gradually increased from 0.4-0.8 M,while the α-helix showed the opposite trend.This indicates that low concentrations of salt ions make the structure more relaxed.With increasing Na Cl concentration,the content of active thiol and carbonyl groups in the myofibrillar protein gradually increased,indicating that Na Cl can cause protein oxidation,and the unfolding of protein structure increases the exposure of amino acids,making them more susceptible to attack by reactive oxygen species.During heating,the content of active thiol and carbonyl groups gradually increased at 85°C and 0-0.4 M,but when the Na Cl concentration exceeded 0.4 M,the content of active thiol groups began to decrease.When the heating temperature exceeded 85°C,the content of active thiol groups significantly decreased with increasing salt concentration,while the content of carbonyl groups sharply decreased at 115°C.This indicates that excessive ion concentration at high temperatures can cause severe aggregation and decomposition of surface active groups of proteins.The results indicate that moderate heating and salt concentration cause the secondary structure to transition from β-sheet to α-helix,making the molecular structure more relaxed and exposing more hydrophobic residues buried inside the molecule,leading to an increase in surface hydrophobicity.The unfolding of the protein structure exposes active sites,making them more susceptible to oxidation.At high temperatures,complex interactions occur between proteins,which exacerbate protein aggregation and oxidation,and may cause burying or even decomposition of active groups.4.Effect of Na Cl on heat-induced myofibrillin digestionThis experiment investigated the effect of Na Cl concentration on protein digestibility under heating conditions using beef myofibrillar protein as the research subject.The results showed that,in the absence of heating,the digestion rate,peptide release,and free amino acid content of myofibrillar protein all increased with the increase of Na Cl concentration from 0 M to 0.8 M.The maximum values were reached at 0.4 M,followed by a decrease,but remained higher than the concentration of 0 M.The effect of Na Cl on protein digestibility was partly due to the increased solubility of myofibrillar protein by salt,and the change in ion concentration caused protein oxidation and denaturation,which destroyed the internal network structure and affected the binding of digestive enzymes to cleavage sites.The digestibility after heating had the same change trend as that of 4℃,and the highest digestibility at 85℃ and 0.4 M was 81.66%.However,a high temperature over 85℃ reduced the promoting effect of Na Cl on digestion.Low concentrations of salt can partially unfold the protein and expose the hydrophobic groups,as supported by the secondary structure changes.Therefore,in the range of 0-0.4 M,it is more conducive to the binding of pepsin,trypsin and specific sites on the protein,so as to promote the digestion of the protein.The changes in the content of binding group,active sulfhydryl and dityrosine found that the high concentration of salt accelerated the rearrangement of myosfibrin,forming more irreversible aggregates through oxidation,especially by the increase of β-sheet content.Mass spectrometry showed that most(> 90%)peptides were 600-2500 Da,and pepsin and trypsin were mainly acting on the myosin heavy chain of myofiber,followed by actin and actin.And most of the restriction sites are α-helical structures,followed by irregular coil,but few βstructures.Therefore,when the Na Cl concentration is 0.4-0.8 M and accompanied by high temperature heating,salting out leads to the reduction of solubility,the degree of oxidative denaturation of protein is too large,which promotes protein aggregation and reduces the α-helical structure,thus burying the specific recognition site of digestive enzyme,resulting in the reduction of protein digestibility.In summary,the synergistic effects of heating and Na Cl can significantly affect protein digestibility,and moderate heating and ion concentrations can effectively enhance protein bioavailability.However,the combined action of excessively high temperatures and ion concentrations can exacerbate protein aggregation and oxidative denaturation,thereby inhibiting protein digestion.In this study,it was found that when Na Cl concentration was 0.4 M and heating temperature was 85℃,the digestion of beef myofibrillar protein was the most beneficial. |
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