Solubilization Of Skeletal Myofibrillar Protein In Water By High Pressure Homogenization And Determination Of Its Novel Functionality

Among various species of proteins,muscle protein is one of the most nutrient-rich food.It has high bio-value,high digestibility,hypoallergenicityand strong flavor,which is considered to be the most excellent protein supplement for the humans.The molecular structure of muscle protein is characrized with long-chain,high flexibility and various of functional groups,thus it can form fluids with different viscoelastic properties whichwould have great potential for the development of texture modified or thicked fluid foods,such as swallowing dietfor elderly people,low-salt and high-protein foodfor special medical purpose,etc.In skeletal muscle protein,myofibrillar protein(MP)is the most abundant component and its functionality largely determines the function,quality and stability characteristics of muscle protein-based products during processing,storage and consumption.However,muscle protein has not been fully utilized to the same extent that plant protein and milk protein hasbeenutilized.This is mainly attributed to the limitation of the fuctional characteristics of MP in aqueous solution at low ionic strength.Solubilization of MP in water,enhacing its thermal stability and improving the functionality of MP powder are important issues that attracted much attention in the field of meat science,and conduction of relevant basic research has become an urgent task.This desertation starts with the innovative introduction of high pressure homogenization(HPH)technology,systematically studies the effect of HPH on the solubility of MP in aqueous solution,and reveals the molecular mechanism of HPH to achieve solubilization of MP in water.Secondly,the effects of different heating temperatures on the solubility,thermal aggregation,rheological properties and molecular properties of water-soluble MP induced by HPH were investigated,and the changes in the thermal characteristics of the MP were revealed.Based on this result,a process strategy of combining HPH with hydrogen peroxide was initiated to suppress MP thennal aggregation so as to improve its thermal stability.In terms of MP powders,this study latterly investigated the effects of HPH treatment on the physicochemical,structural and functional properties of freeze-dried MP powders,and the molecular mechanism on how HPH improved the functional properties of MP powders was clarified.By comparison with the soy protein isolate(SPI)and whey protein isolate(WPI),the composition and techno-functional characteristics of the MP powder modified by HPH were finally revealed.The main findings are shown as followings:1.Potential of high pressure homogenization to solubilize myofibrillar protein in waterThe potential of HPH to solubilize chicken breast MP in water was tested.The effects of 0 psi(0.1 MPa),10,000 psi(69 MPa),15,000 psi(103 MPa)and 20,000 psi(138 MPa)HPH on solubility,protein profile,particle property,flow property and microstructure of MP in water were investigated.HPH at 15,000 psi could induce the suspension of MP with small particle size species(sub-filament,oligomers or monomer structure)and high absolute zeta potential,thus enhancing the solubility,flow ability and stability without individual protein degradation.Reduction of particle size and strengthening of intermolecular electrostatic repulsion appeared to be the main reasons in solubilizing MP in water treated with HPH.The results confirmed that HPH can effectively improve the water solubility and stability of MP.HPH has the advantages of safety,good stability,short treatment time,sterilization,and less influence on nutritional properties which provide great potential when applied indevelopment.of novel muscle protein product.2.Mechanism on solubility enhancement of myofibrillar protein in water related to the comformational change induced by HPHTo clarify the mechanism of solubilization of MP by HPH,we investigated their conformation,solubility and filament forming behavior in low ionic strength solutions induced by 15,000 psi HPH.HPH induced unfolding of MP which subsequently exposed sulfhydryl and hydrophobic groups to the surface.Our findings,determined by circular dichroism,ATR-FTIR,SDS-PAGE and LC-ESI-MS/MS analysis suggested that HPH led to unraveling of helical structures and to formation of myosin oligomers through disulfide bond.Due to intermolecular electrostatic repulsion and physical barrier of disulfide bonds in the rod induced by HPH,we suggested the altered myosin conformation in MP inhibited filament formation,thus contributing to high solubility of MP in water.3.Study on the thermal property of water soluble myofibrillar protein induced by high pressure homogrinizationFor greater utilization of meat as a source of high-quality protein supplements,we investigated the effects of heating(30-90?,10 min)on the solubility,rheological behavior,conformational changes and interactions of HPH-induced watersoluble myofibrillar protein(H-WSMP),in comparison with those of salt soluble myofibrillar proteins(SSMP)and HPH-treated SSMP(H-SSMP).Upon heating above 40?,H-WSMP exhibited a shear-thinning behavior with relatively high solubility and flow ability.The thermal gelling ability appeared to be impaired,probably due to weak myosin-head aggregation(30-50?)and less interaction between myosin tails(70?).H-WSMP was less prone to unfolding during heating,and resulted in smaller protein aggregates,in comparison to SSMP and H-SSMP.Moreover,the lower extent of thermally-induced disulfide cross-links and,hydrophobic and electrostatic interactions led to improved colloidal stability in WSMP.The enhanced solubility and flow ability of WSMP after heating are beneficial to the development of new meat-based products.4.Strategy research on enhancing the thermal stability of myofibrillar protein aqueous solutionAlthough HPH can significantly improve the thermal stability of MP,heavy protein aggregation still exists.Especially when the system has a high protein concentration or the product needs to be subjected to extreme temperature treatment,the heat-induced protein aggregation will be more severe,which is not conducive to the development of muscle protein fluid products having stable and homogenous state.This study tested the potential of high-pressure homogenization(HPH,69 MPa)combined with hydrogen peroxide(H2O2,at 0,40,80,160 and 320 ?mol/g protein)on enhancing the stability of myofibrillar protein(MP,15 mg/mL)against thermal aggregation(90? for 10 min)in aqueous solution.The addition of H2O2 blocked the sulfhydryl groups,inhibited the formation of disulfide bonds,and suppressed thermal aggregation of MP.HPH facilitated the blockage effect of H2O2 by disrupting the intact myofibril structure and exposing buried sulfhydryl groups,leading to a further reduced thermal aggregation and an improved solubility of MP.More than 75%of heated MP remained soluble after the treatment with HPH and 160 ?mol/g H2O2,whereas the control samples formed a gel upon heating.These results suggested that HPH combined with H2O2 is an effective strategy to promote heat stability of MP in the development of muscle protein-based beverages.5.Effect of high pressure homogenization on structural and functional properties of freeze-dried myofibrillar protein powderTo expand utilization of meat in various products,the structural,physicochemical and functional changes of water soluble myofibrillar protein powder(MPP)were investigated as affected by HPH intensities(0-20000 psi).HPH modified the structure of MPP by random dissociation(myofibril and myosin polymer dissociation),partial unfolding and rearrangement(actin trimer formation),producing an amorphous protein structure with high thermal stability.?-helix and ?-turn conversion to(3-sheet structures occurred at pressures above 15000 psi,suggesting an increase in myosin conformation flexibility with minor aggregation.Moreover,HPH was able to improve the water solubility and emulsifying properties of MPP.This might be resulted from its unfolded flexible structure with submicron size and high surface net charge in aqueous suspensions induced by HPH.6.Comparison study on the composition and functionalcharacteristics of myofibrillar proteinpowder modified by high-pressure homogenizationBased on the above studies,this chapter further explored the composition and functional characteristics of HPH-modified myofibrillar protein powder(H-MPP).The compositions(chemical composition,protein composition,amino acid profile and surface hydrophobicity)of the H-MPP were determinedand the functionalities(solubility,water holding and fat absorption capacity,emulsion activity index,emulsion stability index,emulsion droplet size and charge)of H-MPP were compared with those of commonly used proteins(WPI and SPI).H-MPP had high contents of protein(87.40%),which was mainly composed of myosin,actin and tropomyosin.The essential amino acids of H-MPP achieved the FAO/WHO/UNO(2007)standards for pre-school children,and the contents of lysine and sulfur-containing amino acids of H-MPP were higher than those of SPI.H-MPP showed higher surface hydrophobicity while its water solubility was similar to that of SPI,but lower than that of WPI.WSMP demonstrated superior water/oil absorption capacities and emulsifying properties.The fibrous structure and high hydrophobic activity characteristics of H-MPP were able to stabilize oil droplets with submicron droplet size,consequently responsible for its excellent emulsifying properties.The above results can provide a theoretical basis for use of muscle protein powder on the development of novel emulsion-based fluid foods.