Study On Gel Properties And In Vitro Enzymatic Hydrolysis Mechanisms Of Myofibrillar Proteins Based On Oxidation Regulation

Protein oxidative damage is an important reason for the deterioration of raw meat processing performance and quality.In recent years,researchers have focused on how to effectively regulate protein oxidative damage and improve its processing performance.Spices are rich in polyphenols and are often used in meat processing to reduce fat and protein oxidation and improve product flavor.Polyphosphate is a common food additive in meat processing.It has the characteristics of chelating metal ions and dissociating actomyosin,which can regulate the oxidative stability and processing performance of meat protein.Plant polyphenols and polyphosphates often coexist in meat products,but the regulatory mechanism of their combination on the degree of meat protein oxidation and meat product quality is still unclear.In addition,long-term intake of polyphosphates used in traditional meat processing will affect the body’s absorption of calcium.It is of great significance to seek green substitutes for polyphosphates and clarify their mechanisms of action.Therefore,in this paper,the regulation mechanisms of sodium pyrophosphate,catechin and their combination on the structure,oxidative stability,and gel properties of myofibrillar protein(MP)were analyzed by fluorescence spectroscopy,circular dichroism,electrophoresis,scanning electron microscopy and Raman spectroscopy.The in vitro enzymatic hydrolysis mechanism of MP gel was analyzed by infrared spectroscopy,amino acid composition,atomic force microscopy and molecular dynamics simulation.Finally,the possibility and internal mechanism of L-arginine and ε-polylysine as substitutes for sodium pyrophosphate were explored.The main research contents,results,and conclusions are as follows:(1)The effects of different oxidation intensities on the properties of MP gel were investigated under a simulated oxidation system.The results showed that with the increase in H2O2 concentration,the protein carbonyl content increased,and the total sulfhydryl,free amino,and endogenous fluorescence intensities decreased.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDSPAGE)showed that oxidation caused increased protein cross-linking and aggregation,resulting in reduced surface hydrophobicity and solubility.When the concentration of H2O2 was less than 1.0 mmol/L,there was no significant change in the properties of the MP heat-induced gel.When the concentration of H2O2 reached 10.0 mmol/L,the hydrophobicity and solubility decreased by 10.6%and 49.4%,respectively.The cooking loss of heat-induced gel increased by 73.6%,the gel strength decreased by 11.7%,and the gel whiteness did not change significantly.The relationship between protein oxidation strength and gel properties was systematically analyzed,and the moderate and excessive oxidation of proteins were clearly defined.Four representative oxidation strengths were selected,and sodium pyrophosphate was proposed to improve the gel properties of MP with different oxidation damage.(2)The effects of sodium pyrophosphate on the oxidation stability and gel properties of MP under different oxidation intensities were studied.The results showed that protein oxidation induced amino acid side chain modification and protein aggregation,which led to the decline of gel performance,especially at higher oxidation intensity(3～10 mmol/L H2O2).Sodium pyrophosphate can effectively inhibit lipid oxidation in proteins,but its inhibition of protein oxidation is limited.SDS-PAGE analysis showed that when the H2O2 was 0～3 mmol/L,sodium pyrophosphate had no obvious effect on MP protein,but it promoted protein aggregation when the H2O2 was 10 mmol/L.However,sodium pyrophosphate significantly increased the solubility of MP at all oxidation levels.A scanning electron microscope(SEM)observed that the micro structure of MP gel treated with sodium pyrophosphate was more compact and orderly,which was also the reason for the reduction of cooking loss and the increase in gel strength.Due to the limited inhibitory effect of sodium pyrophosphate on protein oxidation,it is proposed to control the oxidative damage of proteins by the synergistic action of catechin and sodium pyrophosphate.(3)The synergistic regulation mechanism of sodium pyrophosphate and catechin on MP structure and gel properties under strong oxidation conditions was studied.The results showed that sodium pyrophosphate and catechin significantly inhibited lipid oxidation and protein carbonyl formation,and their synergistic inhibition was the greatest.Through the determination of particle size,Zeta potential and solubility,the addition of sodium pyrophosphate and sodium pyrophosphate/catechin synergistic samples in the oxidation system reduced the average particle size of MP by 29.7%and 33.5%,respectively,and increased the Zeta potential by 95.4%and 81.6%,and the solubility by 46.9%and 57.6%,respectively,indicating that there was a synergistic effect between the sodium pyrophosphate and catechin.After sodium pyrophosphate treatment,the cooking loss of oxidized MP gel decreased,and the strength of the gel increased significantly.SEM showed that the microstructure of the gel was finer and smoother,similar to that of unoxidized MP gel.Sodium pyrophosphate/catechin improved the gel properties,but the effect of their interaction with MP on the enzymatic hydrolysis of MP gel digestive enzymes still needs to be explored.(4)The effect mechanism of sodium pyrophosphate/catechin on the in vitro enzymatic hydrolysis of oxidatively damaged MP gel was investigated.The results showed that sodium pyrophosphate significantly increased the β-sheet content and pepsin hydrolysis rate of MP gel,while catechin hindered the transformation of ahelix to β-sheet structure and reduced the pepsin hydrolysis rate.Both had no significant effect on the enzymatic hydrolysis rate of trypsin in vitro.Sodium pyrophosphate and catechin significantly improved the antioxidant activity of MP gel digestion products.The hydrolysates(simulated stomach and gastrointestinal stages)of their synergistic treatment chelated 91.4%and 89.1%of Fe2+,respectively,and cleared 59.4%and 77.6%of hydroxyl radicals.All in vitro digestion products contained a large number of small molecular peptides and free amino acids(MW<3 kDa,up to 71.8%).Among them,the content of negatively charged amino acids and the absolute value of Zeta potential in the digestion products treated with sodium pyrophosphate and catechin synergy were the highest,which further confirmed that the presence of sodium pyrophosphate and catechins could significantly improve the antioxidant activity of enzymatic hydrolysates.Since the presence of sodium pyrophosphate and catechin significantly affected the enzymatic hydrolysis of MP gel by pepsin,the interaction between the two and pepsin needs further study.(5)The interaction mechanism between catechin/sodium pyrophosphate and pepsin in vitro was explored.The results showed that catechins competitively inhibited pepsin,while sodium pyrophosphate non-competitively inhibited pepsin.They are reversibly bound to pepsin through non-covalent interactions,causing a decrease in enzyme activity and affecting enzyme catalytic efficiency.Catechin spontaneously combines with pepsin and quenches the intrinsic fluorescence of the enzyme under the control of van der Waals forces and hydrogen bonds;its quenching mechanism is static quenching.Molecular docking and molecular dynamics simulation showed that the most important amino acid site in catechin and pepsin(3pep)is Tyr75,followed by Ser72.The main forces are hydrogen bonding and van der Waals forces,and the number of hydrogen bonds is between 1 and 6.Hydrogen bonds are formed between sodium pyrophosphate and Asn317 of 3pep,and charge interactions are formed between sodium pyrophosphate and Lys319.The number of hydrogen bonds is between 1 and 2.Long-term intake of sodium pyrophosphate will affect the body’s absorption of calcium,and seeking its green alternatives is of great significance.(6)The regulation of L-arginine(L-Arg),a potential substitute for sodium pyrophosphate,on the structure and gel properties of MP under oxidative conditions was studied.The results showed that when L-Arg was at 1～5 mmol/L,it had a certain protective effect on the sulfhydryl group loss,while at 10 mmol/L,the sulfhydryl group decreased by about 7.1%.The addition of L-Arg reduced the fluorescence intensity of endogenous tryptophan,increased particle size,and intensified the cross-linking aggregation of proteins,resulting in a decrease in solubility.With the increased L-Arg addition,the micro structure of MP gel became more compact,but the gel’s strength and whiteness decreased gradually.When LArg was 10 mmol/L,the cooking loss decreased by about 41.5%.5 mmol/L of LArg could improve the oxidative stability and water holding capacity of meat protein,but the gel strength was weak.In view of the similarity of the molecular structure of ε-polylysine(ε-PL)and L-lysine,a potential substitute for sodium pyrophosphate,ε-PL is proposed to replace sodium pyrophosphate.(7)The regulation mechanism of different concentrations of ε-PL on the structure and gel behavior of MP under oxidative conditions was studied.The results showed that the incorporation of ε-PL increased the sulfhydryl content of MP by 9.7%and the solubility by 41.9%,and inhibited protein cross-linking and aggregation.Fluorescence and thermogravimetric analysis(TGA)showed that low concentrations of ε-PL(0.005%～0.020%)promoted the further expansion and instability of MP,while 0.030%ε-PL refolded MP and enhanced its thermal stability.SEM results showed that the physicochemical changes induced by ε-PL were conducive to the formation of a more finer and uniform three-dimensional network structure,which significantly enhanced the gel strength of oxidized MP(increased by 37.7%)and reduced the cooking loss(decreased by 47.1%).Among them,0.020%ε-PL has the best improvement effect.It indicates that s-PL has the potential to replace polyphosphate and can be used to improve the oxidative stability and gel properties of meat products.In this paper,the synergistic regulation mechanism of catechin and sodium pyrophosphate on the oxidative stability and gel properties of myofibrillar protein was studied,and the mechanism of their effects on the in vitro enzymatic hydrolysis of MP gel was revealed.Sodium pyrophosphate increased the pepsin hydrolysis rate of MP gel,mainly because sodium pyrophosphate changed the microstructure of MP gel and the spatial structure of MP,which was basically independent of the interaction between sodium pyrophosphate and pepsin.Catechin reduced the pepsin hydrolysis rate of MP gel.It was due to the change of MP molecular spatial structure and gel microstructure induced by catechin.On the other hand,the catechin-pepsin interaction reduced the catalytic activity of the enzyme.In addition,ε-PL have the potential to replace sodium pyrophosphate.The research results provide a new idea for effectively regulating the oxidative stability and gel properties of meat protein.