Effect Of Pulsed Electric Field Combined With PH Shifting Treatment On The Aggregation Structure And Functional Properties Of Soy Protein Isolate

Soybean protein Isolate(SPI),a kind of a protein product made from defatted soybean meal,has been widely used in different industries such as food,materials and cosmetics for its high nutritional value and functional properties.In recent years,China’s soybean industry has achieved vigorous development and the scale of soy protein production has been expanding.However,when SPI has been extracted and produced,the treatments such as alkali dissolution and acid deposition,heat sterilisation and freeze/spray drying would cause protein aggregation and generate protein aggregates,which results in decreasing solubility of SPI and blocking its functional properties,thus restricting its application in the food industry.So a series of modification methods have been proposed such as by altering the structure of the protein and modulating its aggregation to improve the solubility and functional properties of SPI.However,there are many downsides on the commonly-used methods through thermal,pH shifting and enzymatic modification such as time-consuming processing,energy consumption,involvement of chemical reagents and high cost.Pulsed electric fields(PEF),as a potential new method to replace traditional physical modification methods,has the advantages of short time(μs or ms),low energy consumption and green environmental protection.The present researches of SPI by PEF technology mainly focus to study the effects on its structural and functional properties,but there is still less clear on its mechanism.And early studies found that the modification of plant protein by PEF technology alone proved restricted by high field strength and long-time treatment.The reason for the negative effects also requires to be discussed and studied.In this thesis,in order to solve the problems of unclear mechanism and limited modification effect of PEF technology on SPI.The research is carried out in the following two parts:(1)The effect of PEF on SPI aggregation structure and the mechanism.(2)Dual SPI modification by PEF combined with pH shifting treatment.The study has achieved the regulation of the aggregation structure of SPI,solved the problem of the limited effect of single PEF modification,effectively improved the functional properties of SPI products,and provided theoretical support and technical support for the development and application of highly functional soy protein products in China.The main research contents are as follows:(1)The effect of PEF treatment with different electric field strengths(5-30 k V/cm)on the aggregation state and spatial structure of SPI under neutral conditions(pH 7)was investigated.Treatment at low field strengths(5 k V/cm)did not alter the aggregation structure of the SPI.At moderate field strengths(10 and 15 k V/cm),PEF treatment reduced the turbidity and particle size and increased the solubility of the SPI,with the increase of SPI solubility from 70.16% to 77.35% at 10 k V/cm.The moderate field strength of PEF disrupted the hydrogen bonds and electrostatic interactions between SPI molecules,inducing partial unfolding of the SPI tertiary structure,exposing more negatively charged amino acid residues to the protein surface,and electrostatic repulsion between negative charges weakened the aggregation between protein molecules.However,when the PEF effect was withdrawn,the protein structure was partially reversible and the unfolded SPI tended to revert to the original conformation,resulting in a limited dissociative aggregation.As the electric field strength increased to 20 k V/cm and above,the turbidity and particle size of the SPI solution increased significantly.Protein gel electrophoresis and volume exclusion chromatography results indicated the formation of larger molecular weight soluble aggregates.Insoluble protein aggregates were observed at 30 k V/cm,with solubility reduced to 64.83%.PEF polarization at high field strengths(≥ 20 k V/cm)caused further disrupted the non-covalent forces between SPI molecules,resulting in the further unfolded of the SPI secondary structure,and the unfolding SPI molecules were aggregated through hydrophobic interactions and disulfide bonds,to form high molecular weight soluble and insoluble protein aggregates.Moreover,high field strength of PEF accelerated the oxidation of free sulfhydryl groups of SPI into disulfide bonds,facilitating the formation of protein aggregates.(2)The effect of PEF treatment(5-20 k V/cm)on SPI aggregation structures at different pH conditions was investigated.At pH values far from the isoelectric point(3,9,11),SPI molecules repelled electrostatically due to homogeneous charges,leading to the unfolding of SPI aggregated structures and the dissociation of subunits,with the best depolymerisation occurring at a pH of 11.At pH 11,the SPI exhibited two different aggregation structures after the introduction of PEF treatment.The synergistic effect of moderate field strength(10 k V/cm)and conductivity promoted greater structural unfolding of the SPI,dissociation of more subunits and exposure of more residues of negatively charged amino acids,leading to further SPI disaggregation and dispersion.However,the thermal effect of the solution at higher field strengths(15 & 20 k V/cm)led to re-aggregation of the dissociated and unfolding proteins.The pH 11,10 k V/cm treatment is optimal for inducing a greater degree of unfolding and depolymerisation of the SPI structure without intermolecular aggregation.(3)The effect of the combined PEF and pH shifting treatment on the structural and functional properties of SPI was investigated.The combined treatment(10 k V/cm,pH 11)transformed the SPI structure from the original rigid,compact spherical shape to a loose,disordered “molten spherical state”,and induced the dissociation and re-aggregation of SPI subunits into smaller size soluble aggregates.The value of solubility and turbidity was90.23% and 0.072,respectively,resulting in better aggregation reduction compared to a single pH shifting and other combinations of PEF and pH shifting treatment.Compared to the untreated SPI,the emulsifying activity(EAI),emulsifying stability(ESI),foaming activity(FA)and binding constant to lutein of the combined treated SPI were increased by 119.24%,39.33%,59.03% and 245.81%,respectively.(4)PEF combined with pH shifting treatment was applied to the preparation of SPI nanoparticles,and the loading and protection effect of the nanoparticles on lutein was investigated.Compared to untreated and pH-shifted SPI nanoparticles(SPI7 and SPI11),the SPI nanoparticles(PSPI11)obtained from the combined treatment exhibited the advantages of high surface hydrophobicity,small particle size,uniform dispersion and high stability.When the mass ratio of SPI to lutein was 25:1,the encapsulation efficiency(EE)and loading capacity(LC)of lutein in PSPI11 reached 76.9% and 3.9%,respectively,which were significantly higher than the EE and LC values of the SPI7 and SPI11.The lyophilised powder of SPI-lutein showed good redispersibility and the water solubility of lutein was greatly enhanced,with a 9.5-fold increase in the lyophilised powder of PSPI11(compared to the solubility of lutein crystals in water).In addition,the nanocomplexation with SPIs improved the storage stability(4 °C)and thermal stability of lutein,with PSPI1 offering better thermal protection and a retention lutein rate of 76.4% after 3 h of heating at 85 °C.(5)PEF combined with pH shifting treatment was applied to the modification of commercial SPI,and the effect of this modification method on the solubility and functional properties of commercial SPI was investigated.The PEF treatment had a limited solubilisation effect on the commercial SPI,increasing from only 26.06% to 36.71% of the original at 10 k V/cm field strength.Whereas the combined PEF and pH 11 shifting treatment increased the solubility of the commercial SPI substantially to 70.34%,and the formation of small particle size soluble protein aggregates.The combined modified SPI had higher flexibility,surface hydrophobicity and free sulfhydryl content,and the protein structure tended to be partially folded and disordered,resulting in significantly improved emulsification and foaming performance.The ESI,EAI and FA of the combined modified SPI was increased by 90.05%,34.88% and 104.96%,respectively,compared to the untreated SPI.However,further increase in PEF strength(20 k V/cm)resulted in excessive oxidation and re-aggregation of SPI,reducing the functional properties of SPI.