| Soy protein isolate(SPI)is an important ingredient in many fabricated food systems,but high viscosity is reported to be the main limiting factor of its utilization in food industry.Studies reported that enzymatic hydrolysis improved the functional properties of soy protein isolate,including the reduction of its viscosity.Furthermore,enzymatic hydrolysis also produces polypeptide molecules that are beneficial to human health.However,it is reported that the conventional method is associated with the formation of bitter taste,which is associated with high degree of hydrolysis(DH).Thus,it is assumed that this problem can be solved by adopting the technical solution of limited enzymatic hydrolysis.During the enzymatic hydrolysis,on the other hand,the high viscous nature of soy protein isolate was reported to cause resistant effect of the molecules to bind with the enzymes during enzymatic hydrolysis,leading to low hydrolysis rate and peptide yield.Hence,the use of ultrasound pre-treatment was proposed as the physical shear in the acoustic cavitation could not only facilitate the reduction in viscosity,but also enhance substrate-enzyme bonding and expose the enzyme active new cleavage sites,thus promoting the enzymatic hydrolysis process.Therefore,this study focused on ultrasonic-assisted limited enzymatic hydrolysis technology for reducing viscosity and controlling bitterness of the hydrolysate derived from soybean protein isolate.The following are the main contents and findings of the research:1.Measurement of bitterness levels in soy protein isolate hydrolysate(SPIH)for process control and the development of its prediction models(1)Screening of protease and DH for low bitterness and high peptide yield hydrolysate derived from SPI was performed using five different commercial food-grade proteases(alcalase,bromelain,flavorzyme,papain and protamex)at varying controlled DH(3,6,9,12,15%).The results showed that bitterness of the hydrolysate from protamex,flavorzyme and alcalase proteases increased as DH increased,yet papain and bromelain hydrolysates bitterness showed to decreased after DH reached 9%,where bromelain hydrolysate at DH of 12%was found to exhibit the lowest bitter intensity(p<0.05);at varying DH levels,papain showed to yield the highest peptide content,followed by bromelain.Consequently,proteolysis with bromelain at DH of 12%was found to be the optimal protease and DH to generate low bitter SPIH with high peptide yield.(2)Surface hydrophobicity,relative hydrophobicity and molecular weight distribution of the SPIH prepared using five different proteases at varying controlled DH were observed and their correlation with bitterness was analyzed.The results showed that surface hydrophobicity and relative hydrophobicity decreased with the increase of DH,and the content of small molecular peptides increased with the increase of the degree of hydrolysis;the bitterness of the hydrolysate decreased with the increase of the surface hydrophobicity(p<0.05),yet the correlation between relative hydrophobicity and molecular weight with bitterness showed distinct results between the proteases,where the bitterness of the bromelain hydrolysate decreased with the decrease of relative hydrophobicity and the increase of the molecular weight content less than 3 kDa.(3)Bitterness predictive modelling was developed based on the above mentioned three physicochemical properties,i.e.,surface hydrophobicity,relative hydrophobicity and molecular weight distribution,using machine learning with support vector machine(SVM),linear regression,adaptive boosting(AdaBoost)and K-nearest neighbors(KNN)algorithms.Bitter intensity was assigned as the target,while the three physicochemical properties were assigned as the features.The modelling procedures included data preprocessing with local outlier factor,model development with four different algorithms mentioned above and performance evaluation by 10-fold stratified cross-validation.Single and cross-combination of the features was appointed in the models.Among the models,AdaBoost algorithm models resulted the best prediction performance of bitterness and the root mean square error(RMSE)established by the combination of three physicochemical properties was 0.300 with R2 of 0.979.2.Ultrasound viscosity-reduction and limited enzymatic proteolysis of soy protein isolate(1)Mono-frequency ultrasonic cup was developed by the research group to perform ultrasonic pretreatment on high-density soybean protein isolate.Screening of the ultrasonication condition was conducted with viscosity as the evaluation index.Based on single-factor step-by-step optimization,ultrasonication was found to effectively decrease the viscosity of soy protein isolate at L/S ratio of 14%(w/v)concentration with 20 kHz frequency ultrasound treatment for 10 minutes at 160 W/L power density.Under this concentration level,the unpretreated SPI’s original viscosity was 28067.70 cp;and under the optimized pretreatment condition,its viscosity was reduced by 6.59%(p<0.05).(2)According to the above optimized L/S ratio of 14%(w/v)SPI concentration,screening of the limited enzymatic hydrolysis condition by bromelain to prepare high peptide yield with low viscosity of SPIH was performed.At controlled DH of 12%,the optimal limited enzymatic hydrolysis conditions acquiesced to pH 6.5,temperature of 55℃ and E/S ratio of 7000 U/g.Under these conditions,the peptide yield of the product was 71.496%with the viscosity of 1.676 cp,which was 99.994%lower than that of unpretreated SPI(p<0.05).(3)The effect of ultrasound viscosity-reduction on the preparation of SPIH prepared at controlled DH were measured.Under the optimized ultrasound treatment condition for substrate viscosity reduction,limited enzymatic hydrolysis was performed on the soy protein isolate pretreated by ultrasound with product comprised 77.068%peptide yield and 1.474 cp viscosity,indicating that ultrasonication increased peptide yield by 8.55%(p<0.05)and further decreased the viscosity by 12.05%(p<0.05).3.Effects of ultrasound viscosity-reduction-assisted limited enzymatic hydrolysis of soy protein isolate on the functional properties,bitter intensity and antioxidants activities(1)Functional properties(solubility,emulsifying properties,water-and oil-holding capacity),bitterness and antioxidant activity(OH scavenging activity,DPPH scavenging activity and reducing power)of the hydrolysates were analyzed.The results showed that ultrasonication had no significant effect on the protein solubility,oil-holding capacity and OH scavenging activity of the hydrolysate,but hydrolysates’ emulsifying property,emulsion stability,water-holding capacity,DPPH scavenging activity and reducing power were increased by 17.874%(at pH 7),45.567%(at pH 7),16.60%,13.425%and 23.695%respectively(p<0.05).(2)Bitter intensity was analyzed by e-tongue,sensory analysis and predictive models built in the previous chapter.The results showed that ultrasonication reduced the bitterness of the hydrolysate by 15%(p<0.05);in the utilization of the prediction models,the models built of SVM,AdaBoost and kNN could greatly estimate samples bitter intensity with comparable results with that of sensory analysis.(3)Possible correlation between viscosity with functional properties,bitter intensity and antioxidant activities of the samples were analyzed.Viscosity did not correlate with protein solubility,yet exhibited positive correlation with water-holding capacity,OH scavenging ability and reducing power(R2 of 0.998,1.000 and 0.993 respectively,p<0.05),opposite to emulsifying index,emulsifying stability,oil-holding capacity,DPPH scavenging ability and bitterness(R2 of-0.772,-0.820,-0.996,-0.992 and-0.675 respectively,p<0.05).4.Impact of ultrasound viscosity-reduction-assisted limited enzymatic hydrolysis of soy protein isolate on protein structural properties(1)The effect of ultrasonication on the surface hydrophobicity,relative hydrophobicity,sulfhydryl group,molecular weight distribution,particle size,zeta potential and secondary structure of limited enzymatic hydrolysis of SPI was analyzed.The results showed that ultrasonication reduced the content of small molecular peptides ranged of 1-3 kDa by 3.91%(p<0.05);improved the surface hydrophobicity and relative hydrophobicity(by 2.60-folds and 20.146%respectively);and increased the absorption strength and the fluorescence peak of the hydrolysate,resulting in changes in protein conformation,including:particle size(reduced by 9.837%,p<0.05),sulfhydryl groups(reduced by 12.500%),β-turn(increased by 26.629%,p<0.05),β-sheet(increased by 10.113%,p<0.05),and random coil(increased by 31.946%,p<0.05).(2)The relationship between viscosity,bitterness and protein structure properties was analyzed by Pearson correlation analysis and stepwise multiple regression analysis.The results showed that the reduction of surface hydrophobicity and the increase of β-sheet content were the main reasons for the decrease of viscosity with regression equation of y=1454.389+30.663x1-59.265x2(y,x1,x2 denote viscosity,surface hydrophobicity andβ-sheet content respectively,with R2 of 1.000);meanwhile,the increase of disulfide bond content is the main reason for the decrease of bitterness,with regression equation of y=0.012x6+0.4923x5-8.1327x4+68.732x3-311.55x2+711.67x-619.43(y and x denote bitterness and disulfide bond content respectively,with R2 of 0.764). |
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