Myosin-serum Albumin Complex Gel Formation Mechanism

According to the gel properties of proteins,the properties of myosin and bovine serum albumin mixed in different proportions were thermally induced to form composite gels.The apparent characteristics of the composite gels were studied by water retention,turbidity,solubility,rheology,and scanning electron microscopy.Using ZETA potential,particle size,electrophoresis,circular dichroism,endogenous fluorescence and surface hydrophobicity,the secondary and tertiary structure changes of the composite gel aggregation system were analyzed.The changes of disulfide bonds in composite gels were studied by means of disulfide bonds,diagonal electrophoresis,Raman spectroscopy,and protein mass spectrometry.The following conclusions were drawn,and the final results of the study showed that bovine serum albumin（BSA）could not form a gel with good gel properties at 20 mg·m L^-1.The results of water retention showed that pure myosin gel,myosin∶BSA composite gel15∶5(mg·m L^-1)group,10∶10(mg·m L^-1)group,the water retention results of the three groups were similar,5∶15(mg·m L^-1)decreased water retention.The dynamic rheological results showed that in the process of heating,the storage modulus G’value showed three obvious transitions at 43℃,53℃and 61℃.The G’values of myosin∶BSA composite gel 15∶5（mg·m L-1）group,10∶10（mg·m L-1）group,and 5∶15(mg·m L^-1)group were lower than Pure myosin gel,indicating that the elastic characteristics of the composite gel decreased,and the storage modulus G’was always higher than the loss modulus G’’,indicating that the composite gel formed a more elastic protein gel,adding BSA The tanδvalue of the group was lower than that of the control group at first and then higher than that of pure myosin,indicating that the viscosity ratio of the samples added with BSA increased,which was the same as the conclusion of the storage modulus.Scanning electron microscopy showed that when the myosin∶BSA composite gel was 10∶10(mg·m L^-1),there was cross-linking between the myosin and BSA molecules,the pore size was small and uniform,and a dense and ordered gel network could be formed structure.Turbidity indicates that BSA promotes myosin to form a gel.The increased solubility indicated that the electrostatic repulsion between molecules was greater than the hydrophobic interaction,and the electrostatic repulsion carried by BSA itself promoted the unfolding of the quaternary structure of the complex protein.The ZETA potential showed that the absolute value of the Zeta potential reached a maximum of 6.92 when the myosin∶BSA composite gel was 10∶10(mg·m L^-1),and the electrostatic repulsion involved in the formation of the gel was the largest at this time.New spatial structures are formed under the interaction of force and hydrophobicity.The particle size shows that the average particle size of the composite gel is greatly reduced,and the BSA molecules form a gel with myosin molecules during heat treatment,replacing some of the binding sites between the myosin molecules that form the gel,forming smaller cross-links.body.SDS-PAGE electrophoresis showed that myosin and BSA formed a new protein molecular structure.The new disulfide bonds generated by MHC and MLC are opened under reductive electrophoresis to generate sulfhydryl groups.The results of surface hydrophobicity analysis showed that the main force to maintain the tertiary structure of myosin gel was hydrophobic force,and the main force of myosin to maintain the composite gel after adding BSA changed from hydrophobic force to electrostatic interaction force.Endogenous fluorescence showed that the fluorescence intensity reached the highest at A2,compared with BSA myosin∶BSA composite gel15∶5(mg·m L^-1),10∶10(mg·m L^-1),5∶15(mg·m L^-1)The peaks at 400 nm in each group were blue-shifted,indicating that the emitting groups were located in a more hydrophobic microenvironment.Circular dichroism spectra showed that the percentage of secondary structure content in the myosin∶BSA composite gel 10∶10(mg·m L^-1)group was similar to that in the pure myosin group,the alpha-helix was 46%,the beta-turn was 17%,and the random coil was 24%,with similar gel properties.The total sulfhydryl group and active sulfhydryl group showed that most of the total sulfhydryl group in the composite gel was derived from the myosin molecule,and the overall content of the total sulfhydryl group decreased with the increase of the BSA concentration ratio.The active sulfhydryl groups showed a trend of increasing first and then decreasing.Raman spectroscopy showed that BSA of 10∶10(mg·m L^-1)of myosin∶BSA composite gel combined with myosin molecules to form new disulfide bonds,which constituted a gel with good properties.In the globulin:BSA composite gel group of 10∶10(mg·m L^-1),the exposure of tyrosine residues was the highest,which provided a certain hydrophobic force for the gel structure.Diagonal electrophoresis showed that the proteins or polypeptides contained in A0 and A2 at 220K Da were inferred to be Myosin heavy chain 3 proteins by protein mass spectrometry identification and Uniprot database search,which were connected by intermolecular SS;A0 and A2 contained at 130K Da.The identified protein or polypeptide can be deduced to be Myosin binding protein C2 protein,which is connected by intermolecular SS;the protein that both A0 and A2 show a turning phenomenon at 25～20K Da can be deduced to be two types of Myosin light chain 3,It contains A0A3Q1MBD4 and A0A452DI97,which are eukaryotic reference proteins with the same gene as the center.The two isoforms are different subtypes,and it is not clear that they are connected by intramolecular or intermolecular SS;A0 contains two proteins at50K Da.Or polypeptides are linked by intramolecular SS,containing three proteins or polypeptides linked by intermolecular SS,which can be inferred to be Actin,or other hybrid proteins after identification.A2 contains a protein or polypeptide at 55K Da and50K Da connected by intramolecular SS without intermolecular disulfide bond.After identification,it can be inferred to be Albumin and Actin,or other miscellaneous proteins.Protein mass spectrometry showed that Myosin-2 and Myosin binding protein C2combined with BSA to form an aggregate of the original SS fragment,and formed aggregates with BSA or other myosin molecules to form new macromolecular peptides;in Myosin heavy chain 3 and Myosin binding protein C2 Combined with BSA to generate new SS,which can maintain the stability of the tertiary structure of the composite gel;after heat treatment of myosin and BSA,the SS in the gel is interconverted among different isoforms of the same protein that have not been identified and typed.Myosin-bovine serum albumin composite gel can form a good gel at p H 6.5,10∶10(mg·m L^-1),and the force formed by pure myosin gel is mostly hydrophobic interaction,the force of the composite gel is mostly electrostatic interaction force.The analysis of disulfide bond sites showed that the identified four proteins,Myosin-2,Myosin heavy chain 3,Myosin light chain 3,and Myosin binding protein C2,can bind to BSA or itself,replacing part of myosin to form disulfide bonds.forming gels with good gel properties similar to those of pure myosin gels.This enriches the nutritional properties of gel meat quality and provides a theoretical basis for the ability of BSA to replace meat to a certain extent.