Phase Behavior And Microstructure Of Soy Protein Aggregate-Polysaccharide Mixtures

Phase separation of protein-polysaccharide has been the primary tool for the development of new food processing technologies and design of product microstructure. The development of soy protein is also necessary to recur to this principle to obtain desired food texture attributes.Heat treatment is involved in all soy food processing;as a consequence,the formation of soy protein aggregates is prevalent in all soy foods.So different soy protein aggregates-polysaccharides mixtures were constructed and the phase behaviors and microstructures were studied.The effects of protein concentration,ionic strength,and lyophilization on heat-induced aggregation of soy proteins were analyzed by SDS-PAGE,high performance size exclusion chromatography(SEC-HPLC),laser light scattering(LLS) and zeta potential analyzer.SDS-PAGE profile suggested that the aggregates were formed via non-covalent forces and/or disulfide bonds.At ionic strength of zero, SEC-HPLC revealed that the samples were composed of three major fractions: aggregates,intermediate and non-aggregated molecules.Furthermore,the relative proportion of the aggregate fraction increased from 14.7%to 74.7%as protein concentration increased from 1%to 5%.Similarly,LLS indicated that the average hydrodynamic radius(R_h) increased from 37.02 nm to 144.9 nm.In sample with an ionic strength of zero,the intermediate fraction decreased after freeze-drying with a concomitant increase of the aggregate fraction and R_h.When the sample with concentration of 1%was heated at elevated ionic strength,the SEC-HPLC and LLS profiles changed substantially,the intermediate fractions disappeared,and aggregate fraction and R_h increased markedly;zeta potential result indicated that the charge screening of salt ions accelerated the formation of larger aggregates;and the effect of lyophilization decreased at elevated ionic strength.The mixtures of different soy proteins(native soy protein,1%A and 5%A) withκ-carrageenan(κ-car) were constructed.Phase diagrams were established by centrifugation,chemical assays and visual observation.The microstructures of the phase separated mixtures were described using confocal laser scanning microscopy(CLSM), and image analysis was done.Additional information of microstructures of phase separated mixtures was obtained from small deformation rheology(G',G'').The particle size measurement was performed on the bottom and upper phase of phase separated mixtures.The results of phase diagrams showed that the segregative phase separation took place in the system of soy protein andκ-car,and 5%A/κ-car system had narrower stable region.The observation of CLSM revealed the association of protein-rich areas after phase separation.The results of variance and the histogram of the grey values based on the CLSM images showed that the most heterogeneous microstructure was formed in the 5%A/κ-car system.Rheological measurement results demonstrated that there was higher viscosity of the phase separated system;time sweep indicated that the G'-G" cross-over occurred earlier for 5%A/κ-car mixture.These results amply that the increase of particle size accelerated the phase separation of soy protein withκ-car,and the depletion interaction is the most likely cause of phase separation.Particle size measurement indicated that the larger particles existed at the bottom phase and further evidenced that the phase separation was induced by the depletion interaction.The effects of particle size of soy protein and molecular weight of dextran on the phase behavior and microstructure of soy protein/dextran mixtures were studied by the phase diagrams,CLSM combined with image analysis and rheological analysis.The depletion effect of dextran chain induced the phase separation of two biopolymers,and caused the effective and inhomogeneous association of protein-rich areas.The mixture of dextran with larger molecular weight and larger aggregate phase separated at lower biopolymer concentration.The observations of CLSM demonstrated that gelation completed with phase separation simultaneously and competed with each other.When the phase separation speed was faster than that of gelation,macroscopic phase separation took place;whereas the resulting system was a gel which appeared homogeneous at the macroscopic level,though heterogeneous at the microscopic one and rheological analysis evidenced the formation of the gel.The microstructures of phase separated system of 5%A/dextran at different stage were observed by CLSM,and the results indicated that microstructures evolved with time and agreed with the results of time sweep.The effects of ionic strength on the phase separation of soy protein aggregate/dextran mixtures were studied by the phase diagrams,CLSM and rheological analysis.Adding NaCl to the mixtures of soy protein aggregates/dextran,the repulsion of protein molecules was reduced because of the charge screening of the salt,the larger aggregates formed as a consequence.On the other hand,the phase separation of soy protein aggregates with dextran was also accelerated with the presence of NaCl.The result of phase diagrams confirmed that the increase of aggregate size promoted the phase separation of soy protein aggregate with dextran and outweighed the effect of ionic strength,and further indicated that the depletion interaction plays an important role in the phase separation.CLSM observation showed that the microstructures of the phase separated mixtures depended on the size ratio of polymer to colloid(ξ).Whenξwas small,aggregated-network structure formed while in the mixtures with higherξvalues, the droplet-like structure formed.The rheologcical measurement approved the effects of salt on the soy protein aggregation and phase separation as well.Vrij's depletion interaction theory was used for calculating the theory phase boundaries of soy protein and its aggregates with polysaccharides.The parameters used in the calculation were obtained from static light scattering,and the volume fraction of protein was transformed into concentration by the measurement of relative viscosity in the dilute solution.The theory phase boundaries of native soy protein/1%A/5%A with dextran affinned that with the increase of molecular weight of polysaccharide,the phase boundaries moved to smaller polymer concentrations,and the increase of particle size of colloids accelerated the phase separation.The trends of these results agreed well with the experimental results and suggested that the Vrij's depletion interaction theory is allowed in the description of phase separation of soy protein with dextran.The theory phase boundaries of mixtures of soy protein aggregates and dextran at certain ionic strength lied systematically higher than the experimental results which are ascribed to the neglect of salt effects,since the presence of salt contributed much to the entropy of the system; but at certain protein concentration,the theory phase boundaries corresponded well with the trends of experimental results that the formation of larger aggregates at higher ionic strength accelerated the phase separation.