Investigation Of Protein Physicochemical Properties And Molecular Dynamics To Unveil The Mechanism Of High-pressure Induced Improvements Of Meat Protein Gelation Functionalities

Gel-type meat products are unique in flavor,convenient to eat,and rich in nutrition,and this type of product is an indispensable food in the fast-paced life of contemporary people.In order to meet consumers'demand on high-quality meat products,more and more novel technologies are applied to the processing of gel-type meat products,with the objective to further improve the product quality,making them value-adding products.High-pressure treatment(HPT)is a non-thermal processing technology,as the pretreatment for meat batters,many researches have confirmed that HPT could improve the functional properties,such as texture,water-holding capacity,of meat products.As driven by physical pressure,HPT mainly changes the physical and chemical properties of meat proteins,thereby altering their gelation behavior during heating,finally contribute to the quality modification of meat products.Multiple studies have been carried out to explore the underlying mechanism of HPT-induced modification of meat products in terms of physicochemical properties,these studies fail to reach a highly aligned conclusion due to product variations,HPT parameters,or experimental conditions.Therefore,establishment of a reliable research model system as well as selection of appropriate research methods are the key to build and improve the mechanism of the impacts of HPT on the functional properties of gel-type meat products.In this study,rabbit longissimus dorsi(LD)was used as the raw material.Starting from the meat batter model system,changes of the physicochemical properties of proteins in the rabbit meat batter model system,the microenvironment,as well as the micro structure of the gel were investigated from the perspectives of macroscopic functional characteristics and microscopic protein molecular changes.At this point,the major influences of HPT on the proteins in the model system are preliminary determined.Afterwards,focusing on the myosin model system,investigations on physicochemical properties,molecular morphology,system microenvironment,and gel microstructure of myosin were carried out.Therefore,gaining insights of the underlying molecular mechanism of the quality attributes modifications of gel-type meat products induced by HPT.Finally,computational simulation program was developed to establish a molecular dynamics model to explore the molecular changes under 300 MPa HP condition.Involvements of the major amino acid residues during the process of HPT,changes of chemical force trend,the main driving force of protein unfolding,and the possible action sites of myosin-induced aggregation induced by HPT are the objectives of interest.Based on the results of the physical and chemical experiments and the results of MDS,a mechanistic theory of HPT-induced protein changes contributes to the functional properties was proposed.The major research results of each section are as follows:1.Effect of HPT on the physicochemical properties of meat batter and the macrofunctional properties of thermally-induced gelThe rabbit meat batter was adopted as the model system in this study,which was then subject to HPT at 100-300 MPa for 3,9,or 15 min.Subsequently,the dynamic rheological properties,cooking loss,centrifugal loss,expressible moisture content,textural properties,sensory evaluation of juiciness and chemical interactions of the cooked rabbit meat batters were determined.Results exhibited that HPT severity higher than 200-9(Pressure-times)significantly reduced the cooking loss of the gel,indicating the improvement the yield of final product.Meanwhile,it was also found that the gels in these treatment groups contain higher amounts of expressible moisture content than the other samples,the results of which are similar to the sensory evaluation of juiciness.Determinations of chemical interactions of cooked rabbit meat batters revealed that HPT,especially the samples that were treated with a HPT severity higher than 200-9,has a higher proportion of hydrophobic interactions and hydrogen bonding content.This result accounted for the higher water-holding capacity and better juiciness of HPT samples to a certain extent.The results confirmed that the HPT conditions applied in the current study could exert significant impacts on the functional properties of gel,therefore the reliability of subsequent studies that are supposed to be performed on this model system are guaranteed.2.Effect of HPT on the micro-perspective properties of meat batter and the corresponding gel systemsUsing the above-mentioned rabbit meat batter model system for HPT.Investigations were performed from the perspective of microscopic properties of raw meat batter and the corresponding thermo-gels.Qualitative and quantitative analysis of micro-perspective characteristics,such as microstructure,micro-environment,of cooked rabbit batters were performed.Results shown that when the severity of HPT reached 200-9 or above,the network structure of cooked meat batters has a highly crosslinked three-dimensional structure.Moreover,the microstructure of these treatment groups has a higher fractal dimension value as well as a lower lacunarity.According to the results of principal component analysis,the complexity of the gel network structure(characterized by the fractal dimension)and the porosity(characterized by the lacunarity)does not have a direct correlation with the water-holding capacity,water distribution attributes.Raman spectroscopy analysis exhibited that the gauche-gauche-trans disulfide bond conformation is most stable form under high-pressure condition.Furthermore,HPT induces the rearrangement of the secondary structure of the proteins in the batter system,showing a reduction of a-helix content and the increase of ?-sheet content.The strength of O-H stretching vibration was enhanced in wake of HPT,the results of which are consistent with the increment in the ratio of bound-water,indicating that the gel formed by the minced meat after HPT shown greater water-holding capacity.3.Effect of HPT on the aggregation behavior and physical and chemical properties of myosin moleculesIn this chapter,investigation objectives were centered on perspective of the molecular macroscopic characteristics.Myosin solubility,viscosity of myosin as the function of increasing shear rate,molecular weight,thermally-denaturation behavior during heating,and chemical interactions among molecules were determined.The results showed that the solubility of myosin decreased following HPT;moreover,a tendency to form a protein aggregates was observed by gel permeation chromatography as the mass average molecular weight and number average molecular weight increased in subsequent to HPT,and the molecular weight of the 300 MPa treatment group was doubled compared to the control group.It implies that HPT has a potential to induce myosin mono-molecules to form myosin dimers.In addition,the content of disulfide bond in the samples treated with HPT increased significantly(P<0.05),however,the reducing/non-reducing electrophoresis results showed that the disulfide bond was not the major chemical bond that hold protein aggregates.Analysis of proteolytic fragments by reverse-phase high performance liquid chromatography revealed a new peak at retention times of 5.7-7.5 min,indicating that the formation of myosin aggregates under HPT may follow a specific manner.Conclusively,200 MPa and 300 MPa HPT have the potential to induce a covalent cross-link among myosin molecules stabilize myosin dimers.4.Effects of HPT on the microscopic properties of myosinTo gain comprehensive understanding of the mechanism underlies the HPT-induced modification of myosin,the objective of this chapter took the microscopic perspective of the molecular system.Changes of protein molecular morphology,microstructure of myosin gel,and the microenvironment of myosin model system before and after thermal treatment were determined.Atomic Force Microscopy(AFM)results found that the morphology of myosin molecules treated with 200 MPa or 300 MPa HPT shown a "swelling" phenomenon.Analysis of scanning electron microscopy results revealed that the three-dimensional network formed by myosin treated with 300 MPa has a lower complexity,larger network pores and thicker protein filaments in the gel network structure compared with the control group and the 100 MPa HPT samples.These results are in agreement with the AFM observations.Analysis of Raman spectra revealed that,except for the increase of tyrosine entrapment,no predominant effects of HPT on the micro-environment of myosin solution was obtained.However,significant changes in microenvironment were found in HPT myosin thermo-gels according to Raman spectroscopic analysis.Especially,differences of secondary structures of myosin as reflected by the amide I band,and amide III band were highlighted.Specifically,the de-helical extent in HPT samples was higher than that of the control group following heating,indicating that the potential variations of functional properties of the myosin gels.Furthermore,a more significant effect of HPT on the microenvironment of the myosin system appeared after gelation,which indicates there might be a "intermediate state" of myosin induced following HPT.Consequently,changes of myosin "intermediate state" would be magnified by thermal treatment,promoting the divergence of physicochemical properties among myosin molecules that induced by HPT.5.Investigation of the mechanism of myosin monomer structural changes under HPT by molecular dynamics simulationTo unravel the mechanism of myosin changes under HPT,the proposal of‘water penetration' was suggested to explain the myosin structural changes under HP condition(i.e.300 MPa in the current study).To verify the hypothesis,molecular dynamics simulation(MDS)was programmed to explore the underlying details of myosin molecular changes(Protein Data Bank ID:5H53)under 300 MPa.Cyclical water-insertion was applied to the myosin crystalline till saturation,followed by a 45 ns MDS of pressure-holding period.Results of root-mean-square-deviation and root-mean-square-fluctuation values increased under HP during water insertion cycles;solvent-accessible-surface-area was increased following water insertion under 300 MPa,indicating that myosin structure was unfolded as a function of water insertions under HP condition.Repulsive forces between water and hydrophobic residues,especially those located at 100-160,200-245 and 550-650 regions,accounted largely for the molecular unfolding.A slight reverse trend of structural changes was observed during 45 ns MDS,however the overall alteration trend of exposed hydrophobic sites and reduced a-helix content remained.The hypothesis of 'HP-induced water penetration' was validated as the major factor that contributes to protein structural changes.6.Investigation of the mechanism of structural changes of myosin aggregate under HP condition by MDSBased on the results of MDS single molecular system,a two-molecule MDS model was established using the myosin conformation after HP-induced water penetration.To investigate the formation of myosin dimer under HP condition as well as the myosin complex structural changes as well as the alterations of important chemical forces under HP condition.Results obtained from the multi-molecules MDS showed that the tertiary conformation of myosin complex stabilized gradually under the 300 MPa HP condition,and the values of the radius gyration and SASA of the protein complex further decreased with the extension of the simulation time.These results indicate that myosin conformational tightness increased with the dwelling time extending.In addition,the hydrophobic groups at the interface of myosin complex are continuously exposed during the conformation stabilization process.As a result,the increased interaction between protein molecules enables the formation of hydrogen bonds between proteins.As the adductive function of all these chemical forces,the conformation of the protein complex is further stabilized.Although the formation of a-helix between/within the protein molecules after the docking of myosin molecule,the overall trend of secondary structure changes is remained,manifested as the decrease in ?-helix content and relatively increased content of other secondary structures.In summary,from the perspective of MDS,at the initial stage of HPT,myosin molecules form dimers through hydrophobic interactions as driven by ultrahigh pressure.With the extension of pressure holding times,the structure of myosin complexes is further compressed,along with the exposure of more hydrophobic regions,formation of hydrogen bonds between molecules.The overall changes of myosin complex develop in the direction of stabilizing the molecular conformation.In conclusion,changes of myosin physicochemical and functional properties under HP condition are driven by "water penetration" as promoted by ultra-high pressure.Consequently,repulsive interactions between water molecules and hydrophobic residues in the myosin promote the unfolding of molecule,destroying the hydrogen bonds,thus reducing ?-helix content of the protein.Differences in HP intensity and HP holding times would lead to the variation in equilibrium state of myosin molecules.Therefore,the physical and chemical properties of myosin are modified,forming different threedimensional gel network upon thermal gelation process.All these modifications induced by HPT finally contribute to the differences in the functional characteristics of protein gel.