Effects Of Protein Behaviors On Dough Rheological Properties Of Wheat Varieties In Flour Mixing

Quality improvement is a significant objective in wheat breeding.Exploring wheat protein movement and integration during dough development is essential to breed wheat cultivars to meet the requirements of various end-products.In this study,a response to extraction-system was used for the detection of wheat protein behaviors during dough processing and was built on the integrated utilization of SE-HPLC and 2-DGE.Using the methodology that was developed,dough samples from two pairs of wheat cultivars with different Mixolab parameters were investigated to elucidate their differential protein behaviours under continuous mixing and thermal treatment.As for the two paired cultivars,Westonia and Wyalkatchem are distinguished by their poor and good noodle-making quality,respectively;Ventura-26(26% amylose)and Ventura-19(19% amylose)are NILs where the former shows higher amylose content.The comparison of protein behaviors of the four cultivars during dough development provided valuable information for clarifying the association between protein behaviors and dough rheologial properties.The main results obtained in this study as follows:1.The response to extraction-system method focussed on the tracking and detection of dough protein dynamics was establishedDough protein fractions were quantified by SE-HPLC firstly to clarify their changes during dough development.The non-gluten and gluten proteins in dough matrix were sequentially separated by 0.5 M NaCl(pH 7.0)and SD(0.3% SDS,15 mM DTT)for 2-DGE analysis.Protein extraction buffer UCD(8 M Urea,4% CHAP and 60 mM DTT)was used for the separation of total proteins.All the differential proteins were identified by MS/MS finally.2.32 min(80 ?)is a critical time point in terms of forming large protein complexesOverall dough protein extractability decreased dramatically after 32 min mainly due to the extractability loss of y-type high molecular weight gluteinin subunits(HMW-GSs),gamma-gliadins,beta-amylases,serpins,and some metabolic proteins with higher mass.These proteins prone to integrated into the protein polymers in dough matrix and showed reduced extractablity.3.Dough viscosity at C4/38 min(85 ?)was influenced by different mixtures of protein aggregation/disaggregation in dough matrixFor the two cultivars with different noodle-making quality,Westonia showed higher protein extractability compared to Wyalkatchem at early stages of dough mixing(up to 32 min;80 ?)although it was opposite fashion thereafter.The dough torque of Westonia rebounded within short time due to the additional proteins,gamma-gliadin,globulin-1,peroxidase and enolase-like proteins,that were extracted from Westonia?s dough at C3/32 min(80 ?)being repolymerised into the dough matrix at C4/38 min(85 ?);while compared to Westonia,more proteins,alpha/beta-gliadin,globulin-3,globulin-1 and LMW-GSs,were depolymerised from the dough of Wyalkatchem at C4/38 min(85 ?)and no protein was repolymerised into dough matrix,leading to the sharp drop on its Mixolab curve until 43 min.Thirty-three differentially expressed proteins between Westonia and Wyalkatchem could be mainly assigned to chromosomes 1D,3A,4A,4B,4D,6A,6B,7A and 7B.4.The protein-starch interaction in dough matrix was affected by starch compositionsFor the NILs differ in amylose content,significant differnces on the protein behaviors were found between Ventura-26 and Ventura-19 before and after the starch pasting.Stress associated and metabolic proteins largely interacted with dough matrix of Ventura-26 after 26 min(56 ?);gliadins,avenin-like b proteins,LMW-GSs,and partial globulins showed stronger interactions within the dough matrix of Ventura-26 at 32 min/C3(80 ?),thereafter,however,stronger protein interactions were observed within the dough matrix of Ventura-19 at 38 min/C4(85 ?)and 43 min(80 ?).Fourty-six proteins associated with changes in dough matrix due to reduced amylose content were identified by mass spectrometry and mainly annotated to the chromosome group 1,4,and 6.5.The specific proteins related to dough rheological properties were classified as three groups according to their behavioural characteristics during dough developmentA total of 92 differentially expressed proteins from the two paired wheat cultivars were identified according to their extractability variations among different cultivars or different stages during dough development.The proteins were mainly classified as storage proteins(49.68%),stress/defend related proteins(24.35%)and proteins involved into carbohydrate metabolisms(12.10%).Out of them,48 differential proteins appeared at the early stages during dough development showed limited affection on the dough rheological properties,but 44 differential proteins identified from the starch pasting stages were involved into the protein aggregation/disaggregation and protein-starch interactions.Eighteen proteins rich in albumins,gliadins,ALPs and some enzymes possessed both the aggregation/disaggregation and proteinstarch interaction activity,affecting dough rheological property dramatically.Nine gluten proteins including HMW-GSs,LMW-GSs and some ?/?-gliadins were degraded partially during starch pasting,they have the potential to generate larger but weaker polymers with other proteins,which stabling dough strength to a certain extent.Seventeen proteins involve in stress/defend and metabolisms showed high affinity with starch,having the potential to affect dough rheological property via mediating the protein-starch interaction.6.Gene expression characteristics prediction for the identified proteins related to dough rheological propertiesMost proteins related to dough rheological properties expressed in wheat kernel at different layers during grain filling period.According the prediction for some key proteins,such as globulin,gliadin,ALPs and stress related proteins,wheat processing quality would be expected to improve via selecting cultivars with different grain filling efficiency,nitrogen or sulphur application and flour yield adjustment.Based on the optimization of dough protein extractive process,a response to extractionsystem using SE-HPLC,2-DGE and MS/MS was developed for the extraction,separation and identification of dough proteins during flour mixing.A series of differential dough proteins involved into protein aggregation/disaggregation or protein-starch interactions were identified from the four cultivars differ in processing quality.Out of them,the sequential behavioral dynamics of 44 proteins separated from the starch pasting stages were proposed and subsequently,the gene expression characteristics for some key proteins related to dough rheological properties was predicted.The findings expanded our traditional knowledge about wheat quality related proteins,providing theoretical basis for improving wheat quality at cultivation,breeding and processing level.