Study On The Preparation Of Myofibrillar Protein-Dextran-Polyphenol Covalent Complex And The Mechanism Of Stabilizing Emulsion

Meat,playing an important role in the dietary structures of people,can provide high-quality proteins and rich nutrients for the human bodies.Myofibrillar protein（MP）is the most abundant and important protein in the muscle,and its functional properties directly affect the final qualities of the meat and meat products.In the processing of gelled-emulsified type meat products,various additives were added to enhance the qualities of products and the acceptance of consumers.In recent years,the interactions between various additives such as carbohydrates,polyphenols,transglutaminase,non-meat proteins and MP to improve the processing characteristics and sensory qualities of the gelled-emulsified type meat products have been extensively studied by the domestic and foreign researchers.Among the additives,polysaccharides and polyphenols are two important biomolecules,and the mechanism of their non-covalent and covalent modifications with MP has been fully elucidated.However,the molecular mechanism of the synergistic covalent modification of MP by polysaccharides and polyphenols and the influence on the functional properties of MP have not yet been revealed.Based on this,the purpose of the present study was to first prepare MP-polysaccharide-polyphenol ternary covalent complexes,explore the molecular mechanism of the synergistic covalent modification of MP with polysaccharides and polyphenols,and further analyze the influence of synergistic covalent modification of polysaccharides and polyphenols on molecular conformation of MP.Then,the mechanism of synergistic covalent modification of polysaccharides and polyphenols to improve the functional properties of MP were revealed.On the basis of this,the mechanism of MP-polysaccharide-polyphenol ternary covalent complexes stabilizing emulsions containing unsaturated fatty acids was further systematically elucidated.The research results can lay a sound theoretical basis for the synergistic modification of protein functional properties by polysaccharides and polyphenols,and provide important academic significances and application values for expanding the use of muscle proteins in the food industry such as building delivery systems for embedding bioactive substances.The specific contents and results of the study are as follows:1.Preparation and characterization of MP-dextran covalent complexIn order to explore the feasibility of using mild wet-heating reaction to achieve the covalent binding of dextran to MP,MP was reacted with different molecular weights of dextran（20 k Da,40 k Da and 70 k Da）at 37°C for 8 h in the present study.and the covalent binding of dextran with different molecular weights to MP was proved by UV-Vis spectrophotometry,SDS-PAGE electrophoresis and trinitrobenzene sulfonic acid（TNBS）method;The MP that was simply heated at 37°C was used as the control group to investigate the effect of covalent modification of dextran on the turbidity,particle size distribution,rheological properties and microscopic morphology of MP.The UV absorption value of the reacted samples solution and the results of SDS-PAGE electrophoresis showed that different molecular weights of dextran and MP had reacted under the experimental conditions,and a colorless intermediate product was formed.The results indicated that with increase of the molecular weight of dextran,the grafting degree of glycation increased significantly（P<0.05）.When the molecular weight of dextran was 70 k Da,the grafting degree of glycation was 8.8%.The turbidity and fluidity of glycated MP solution were significantly increased（P<0.05）,and the interaction between the protein molecules was inhibited.In addition,with increase of the molecular weight of dextran,the particle size of glycated MP decreased significantly（P<0.05）,and the distribution of MP molecules in the solution was much more uniform.Therefore,the above results indicate that the MP-dextran covalent complex could be prepared by the mildly wet-heating reaction,and the regulation of MP properties by the glycation was affected by the molecular weight of dextran.The grafting degree of glycation was highest when the molecular weight of dextran was 70 k Da.2.The mechanism of dextran conjugation improving the functional properties of MPOn the basis of the above research results,changes in the secondary and tertiary structures of MP after glycation were mainly analyzed to elucidate the mechanism of dextran conjugation improving the functional properties of MP,and the effects of three different molecular weights of dextran covalent modification on the solubility and emulsifying properties of MP were further investigated.The results showed that the covalent modification of dextran had a significant effect on the molecular conformations of MP.Theα-helix contents of MP was significantly reduced（P<0.05）after the glycation,and the side chain groups of aromatic amino acids were exposed to the surface of the protein molecules.And with increase of the molecular weight of dextran,the surface hydrophobicity and Ca²⁺-ATPase activity of glycated MP were significantly decreased（P<0.05）.Compared to the untreated MP and MP heated at 37°C,the emulsification activity（EAI）and emulsion stability（ESI）of glycated MP significantly increased（P<0.05）with increase of the molecular weight of dextran.Furthermore,the solubility of MP at the isoelectric point（p H 5.5）and reaction conditions（p H 7.5）were significantly increased（P<0.05）after glycation.The solubility of glycated MP at p H 7.5 was 88.5%when the molecular weight of dextran was 70 k Da.In summary,the covalent binding of dextran increased the hydrophilicity of MP molecules and resulted in unfolding of the MP molecular conformation.Moreover,the steric hindrance effect generated from the covalent binding of dextran molecules improved the dispersibility and stability of MP in the solution,leading to improving the solubility and emulsifying properties of MP.3.The effect of polyphenols conjugation on the thermal stability and antioxidant properties of glycated MPIn order to further clarify the effect of polyphenol conjugation on the thermal stability and antioxidant properties of glycated MP（MP-dextran covalent complex）,epigallocatechin gallate（EGCG）,catechin（C）and gallic acid（GA）were used to prepare the MP-dextran-polyphenol ternary covalent complexes by radical grafting method.Changes in the molecular conformation of the side chain amino acid groups,secondary and tertiary structure of MP-dextran-polyphenol ternary covalent complex were determined using untreated MP and glycated MP as the control groups.And the intrinsic correlation between the molecular conformation,thermal stability and antioxidant properties of the MP-dextran-polyphenol ternary covalent complexes were established.The results showed that the contents of EGCG,C and GA in the ternary covalent complexes were 58.02μmol/g,44.63μmol/g and 21.97μmol/g,respectively.The free amino,sulfhydryl and tyrosine groups in the glycated MP were participated in the covalent grafting reaction with polyphenols by compared with the untreated MP and glycated MP.The covalent binding of the EGCG,C and GA led to the further unfolding of the glycated MP,and the hydrophobic amino acid groups exposed to the surface of protein molecules.The covalent binding of the EGCG,C and GA inhibited the unfolding and cross-linking of the myosin head,and the thermal stability and denaturation temperature of the glycated MP were significantly increased（P<0.05）.In addition,with increase of the phenolic hydroxyl contents in the polyphenols molecular structure,the free radical scavenging ability of the MP-dextran-polyphenol ternary covalent complexes was significantly improved（P<0.05）,and the free radical scavenging ability of MP-dextran-EGCG covalent complex was highest.Overall,the conjugation of the EGCG,C and GA enhanced the thermal stability and antioxidant properties of the MP-dextran complex.4.Preparation and characterization of flaxseed oil emulsion stabilized by MP-dextran-polyphenol covalent complexIn order to evaluate the effect of MP-dextran-polyphenol ternary covalent complex in stabilizing the emulsion system containing unsaturated fatty acids,the untreated MP,glycated MP and MP-dextran-polyphenol ternary covalent complex stabilized flaxseed oil emulsion systems were firstly prepared in the present chapter,and changes in the particle size,rheological properties,flow properties and in vitro simulated digestion properties were compared.The results showed that the covalent binding of dextran and polyphenols significantly decreased（P<0.05）the particle size of MP-stabilized emulsion system comparing with the untreated MP-stabilized emulsion,and the MP-dextran-polyphenol ternary covalent complex-stabilized flaxseed oil emulsion showed the smallest particle size.The results of viscosity curve analysis of the emulsion showed that the apparent viscosity of the emulsion stabilized by the MP-dextran-polyphenol ternary covalent complex was the highest,and the apparent viscosity of the emulsion increased with increase of the molecular weight of polyphenol.The results of ¹H high-field nuclear magnetic resonance analysis also proved that the interaction between protein molecules and oil molecules in the emulsion stabilized by MP-dextran-polyphenol covalent complexes enhanced compared with that of the emulsion stabilized by the untreated MP and glycated MP,and the mobility of water molecules reduced.After simulated gastric digestion in vitro,compared with the untreated MP-stabilized emulsion,the emulsions stabilized by the glycated MP and MP-dextran-polyphenol covalent complexes shoed lower particle size values during the gastrointestinal digestion stage（P<0.05）;in addition,the MP-dextran-EGCG ternary covalent complex-stabilized flaxseed oil emulsion showed the lowest free fatty acid release rate in the simulated intestinal digestion stage in vitro,indicating the covalent binding of dextran and polyphenols could enhance the stability of MP-stabilized unsaturated fatty acid emulsion system during simulated gastrointestinal digestion in vitro.5.Study of the mechanism of MP-dextran-polyphenol covalent complex stabilizing the flaxseed oil emulsionOn the basis of the above research results,in order to study the mechanism of MP-dextran-polyphenol covalent complex stabilizing the flaxseed oil emulsion,the changes in the protein concentrations,contents and molecular conformations of MP-dextran-polyphenol ternary covalent complexes at the oil/water interface were analyzed.The results showed that the covalent conjugation of dextran and polyphenols significantly reduced（P<0.05）the creaming index of flaxseed oil emulsion during the storage compared with that of the untreated MP-stabilized emulsion.In addition,the lipid hydroperoxide（primary product of fat oxidation）,TBARS（secondary product of fat oxidation）and carbonyl contents of MP-dextran-polyphenol ternary covalent complexes-stabilized flaxseed oil emulsion were significantly lower（P<0.05）than those of the untreated MP and the glycated MP-stabilized emulsions after 7 days storage;it was found that with increase of the emulsion storage time,the protein thiol contents of all emulsions significantly increased（P<0.05）by measuring changes of the protein thiol contents of the emulsion systems after 7 days storage.The thiol contents in the flaxseed oil emulsion stabilized by MP-dextran-polyphenol ternary covalent complex were higher than that of other emulsion systems when the storage time was up to 7 days.It was found that the protein contents and concentrations of MP-dextran-polyphenol ternary covalent complexes at the oil/water interface increased significantly（P<0.05）by measuring the interface protein contents and concentrations.The interfacial protein concentration of the MP-dextran-EGCG covalent complex-stabilized flaxseed oil emulsion was the highest,which was 9.52 mg/m²;in addition,it was found that the covalent grafting of polyphenols promoted the glycated MP unfolding and rearrangement at the oil/water interface,resulting in the formation of a much more stable interfacial protein film by further analyzing the molecular conformational changes of the interfacial protein molecules.Overall,the MP-dextran-polyphenol covalent complex improved the physical and chemical stability of flaxseed oil emulsion.In conclusion,MP-dextran-polyphenol ternary covalent complexes were prepared by mild wet-heating glycation and free radical grafting method.The covalent binding of dextran and polyphenols enhanced the functional properties of MP.The structure and functional properties of MP-dextran-polyphenol ternary covalent complexes were affected by the molecular weights and types of dextran and polyphenols.In addition,the flaxseed oil emulsion stabilized by the MP-dextran-polyphenol ternary covalent complexes showed excellent physicochemical stability,which will provide a theoretical foundation for the establishment of a delivery system for delivering healthy polyunsaturated fatty acids using MP-dextran-polyphenol ternary covalent complexes as the matrix,and provide a new way for the further application of muscle proteins in the food industry.