| Sunflower is among the top five important oilseed crops in the world.China ranks fifth(2.58 million tonnes in 2017),accounting for 5.40%of global production scale.Huge quantity of sunflower meal(0.981 million tonnes in 2017)is being produced,containing 300-500 gprotein/kgmeal.This protein can be extracted for feed and/or food purposes or it can be hydrolyzed to peptides for food,cosmetic and pharmaceutical applications.Recently,researchers have examined the application of novel technologies including microwave,pulsed electric field and ultrasound in extraction and enzymolysis processes to overcome the shortcomings of classical methods(such as high cost,low extraction yield and low enzyme activity).Among them,ultrasonication attracts great attention due to its mild effect on protein for enhancing the safety and quality of treated food.Based on this,this work intensely investigated the impacts of ultrasound-assisted sunflower protein extraction and enzymolysis,and discussed its influence on physico-chemical,techno-functional and structure attributes as well as the antioxidative potential of sunflower protein and its hydrolysatesThe main research contents and findings are as follows:1-The influence of ultrasound-assisted protein extraction from sunflower meal was optimized.Optimization of experimental conditions was achieved to investigate the effect of power density,temperature and sonication time on protein yield and energy consumed during extraction process with Box-Behnken's design.The optimal extraction conditions were observed at a power density of 220 W/L,temperature of 45? and extraction time of 15 min Under optimum sonication conditions,the measured yield of protein and energy consumed values were 54.26%and 0.13 kW.h,respectively.The observed values for these responses were found to be comparable to the predicted values obtained by the mathematical models Physicochemical and functional attributes of protein isolate were also examined at the optimal points.Protein purity,colour lightness,bulk density and oil holding capacity of sonicated sample increased by 2.16,6.97,8.60 and 24.27%,respectively,while particle size and water holding capacity reduced by 31.98 and 18.18%compared with control.The highest solubility,emulsifying activity and emulsion stability were 74.59,52.45,and 50.45%respectively and were obtained at pH 9.0 following sonication2-The effect of dual-frequency ultrasonic(DFU)pretreatment on enzymolysis/proteolysis kinetics and thermodynamics of sunflower meal protein(SMP)using alcalase was examined.The results indicated that DFU pretreatment enhanced SMP enzymolysis efficiency compared with control at different substrate and enzyme concentrations,temperatures and pH.Kinetics analysis indicated that DFU pretreatment reduced the Michaelis constant(km)value by 11.29%,while the apparent breakdown rate constant(kA)value increased by 1.96%,indicating DFU pretreatment improved the affinity among SMP and alcalase.Additionally,the values of reaction rate constant(k)for DFU pretreatment were increased noticeably by 45.96,26.92,21.14 and 27.89%at 293,303,313 and 323 K,respectively(P<0.05).Regarding thermodynamic parameters,DFU action significantly(P<0.05)reduced activation energy(Ea),enthalpy(?H)and entropy(?S)by 24.28,26.13 and 9.10%,respectively.Meanwhile,DFU had a slight influence on Gibbs free energy(?G)when the temperature increased from 293-323 K.3-Proteolysis kinetics and structure attributes of SMP pretreated using sonication of frequency type single(20,40 kHz)and dual(20/40 kHz)were studied.A simplified model with impeded enzyme reaction was developed and the hydrolysis of SMP in a heterogeneous system was successfully described.Initial observed rate(kin)increased after sonication,demonstrating the impacts of sonication on SMP by enhancing proteolysis and altering the structure of SMP,which was validated by circular dichroism(CD)spectroscopy,ultraviolet-visible(UV-Vis)spectroscopy,scanning electron microscopy(SEM),atomic force microscopy(AFM),Fourier transform infrared(FTIR)spectroscopy and sodium dodecyl sulfide polyacrylamide gel electrophoresis(SDS-PAGE)analyses.From SMP characterization,there was a reduction in ?-helix,enhancement in absorption intensity and alterations in functional clusters of SMP following sonication.SEM and AFM observations indicated that sonicated SMP became loosed and exhibited more heterogeneous structure,and irregular small-sized particles than control.SDS-PAGE profile displayed notable changes in molecular weight after sonication,especially for 20/40 kHz,evidenced limited unfolding in SMP conformation.4-Functionalities,conformational characteristics and antioxidative capacities of SMP and its hydrolysates(SMPH)at various degrees of hydrolysis(6-24%)were investigated following sonication.Enzymolysis notably enhanced the solubility,foaming properties and emulsion stability index of untreated and sonicated SMP at most examined pH.Nonetheless,emulsion activity index of SMP was more than SMPH at all pH values,especially at pH 4.0-10.0(P<0.05).Compared with control,sonication improved solubility,foaming capacity and emulsification properties,but decreased foaming stability and had a significant influence on the SMP and SMPH structure.Furthermore,sonication efficaciously enhanced reducing power and superoxide and ABTS radical scavenging capacity of all preparations(P<0.05)over control,confirmed by the analyses of hydrophobicity and content of amino acid compositions.5-Impacts of localized/controlled enzymolysis and sonication on physical,techno-functional,and structure attributes of SMP and SMPH were studied.SMP was subjected to enzymolysis(using alcalase)to prepare SMPH with various degrees of hydrolysis(6-24%DH).Enzymolysis decreased colour lightness,turbidity,and particle size of unsonicated and sonicated SMP,while it increased the absolute values of zeta potential(P<0.05).Sonication improved oil absorption capacity and dispersibility over unsonicated samples.Contrarily,sonicated preparations showed a decrease in water holding capacity.Intrinsic fluorescence and FTIR spectral analyses suggested that SMPH had more movable/flexible secondary structures than SMP.Moreover,the changes in sulfhydryl clusters and disulfide linkages following sonication demonstrated limited unfolding of SMP and SMPH structure and decrease in intermolecular interactions.SDS-PAGE profile exhibited a significant reduction in molecular weight(MW)of sonicated SMP,whereas did not display differences between unsonicated and sonicated SMPH.From further MW analysis,SMPH was categorized with high proportion of small-sized peptides ?3 kDa fractions,which increased from 78.64%to 93.01%(control)and from 82.3%to 93.88%(sonication)with enzymolysis(6-24DH).6-Ultrasound pretreatments of SMP to yield high antioxidant capacity from its hydrolysates by response surface methodology was optimized.Optimization of experimental conditions was achieved to examine the impact of temperature,solvent-solid ratio and sonication time on antioxidant capacities of SMP hydrolysates with Box-Behnken's design.Quadratic models of DPPH-scavenging activity(DPPHSA),hydroxyl-radical scavenging activity(HRSA)and Cu2+and Fe2+chelating activity(Cu2+-CA and Fe2+-CA)were developed,and their coefficients were observed from the multiple-regression analysis.ANOVA indicated that time was highly significant(P<0.01)on all experimental responses.The best experimental condition of DPPHSA,HRSA,Cu2+-CA and Fe2+-CA was accessed at 42.50?,18.16 mL/g and 26.52 min,and the observed data for these responses were 49.92,71.83,51.02 and 44.87%,respectively.The outcome of the verification experiment was reliable with predicted data for all responses.Additionally,DPPHSA,HRSA,Cu2+-CA and Fe2+-CA of pretreated hydrolysate improved(P<0.05)by 17.41,20.00,14.72 and 26.41%,respectively over nonsonicated hydrolysate.Amino acid content and hydrophobicity of SMP hydrolysate at the optimum sonication condition were analyzed.Analyses indicated that ultrasonication could facilitate the releasing of hydrophobic amino acids from SMP over nonsonicated samples during enzymolysis with high antioxidative capacity. |
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