Microstructure formation and rheological behaviour of dough under simple shear flow

In a z-blade mixer, both shear and extensional deformations contribute to the development of dough structure. I effect of simple shearing versus z-blade mixing at similar levels of work input, on the microstructure and uniaxial extensional properties of two doughs prepared from flours of different strengths. With respect to microstructure, mixing initially increased the formation of coarse protein patches, leading to a heterogeneous dough structure with a high fracture stress (σ_max) and significant strain hardening. These parameters decreased with prolonged mixing. This was accompanied by loss of glutenin macro polymer (GMP) wet weight and formation of a more homogenous microstructure. Prolonged mixing typically led to an over-mixed state. In contrast, prolonged simple shearing did not affect GMP content or strain hardening and gave enhanced shear banding. Confocal scanning laser microscopy revealed that short-term simple shearing induced structure formation in the direction of the shear flow for both flour types, followed by formation of shear-banded gluten structures both parallel and perpendicular to the direction of shear flow. Uniaxial extension of dough oriented parallel or perpendicular to the shear field did not reveal anisotropy. Apparently, the observed heterogeneity on a scale of ‘mm’ was not relevant for this type of rheology. Nevertheless, a relative weakening of dough strength (reduced fracture stress) was observed as a function of long-term shearing. This seems to be related to a local segregation effect caused by differences in visco-elasticity between the gluten phase and the starch granules. The results of this study reveal important features of the dough processing and underline the importance of not only work input, but also the type of deformation applied.     

The effect of mixing on glutenin particle properties: aggregation factors that affect gluten function in dough

Previously we reported that the SDS insoluble gel-layer: the Glutenin Macro Polymer (GMP) can be considered as a gel consisting of protein particles. These glutenin particles have a size of about 10⁻¹–10² μm and consist of HMW-GS and LMW-GS only. In GMP isolates from flour, the particles are spherical. In isolates from dough, glutenin particles have lost this shape. This seems relevant, since mixing disrupts the particles and the mixing energy required for dough development correlated with the glutenin particle size in flour. The question studied in this paper is how changes at a glutenin particle level affected the subsequent process of gluten network formation during dough rest and if this could be used to explain resulting dough rheological properties. To this end, we studied how various mixing regimes affected the dough properties during and after resting (elasticity). We cannot fully explain the differences in the final dough properties observed using parameters such as the quantity of GMP in flour, the quantity of re-assembled GMP in dough and the size of re-assembled glutenin particles. However, other parameters were found to be important: (1) the Huggins constant K′ reflecting the tendency of glutenin particles to interact at level II of the Hyperaggregation model; (2) the composition of glutenin particles affecting the potential to form smaller or larger particles and (3) for over-mixed dough, covalent re-polymerisation at the so-called level I of hyperaggregation. Using these parameters we can better explain dough viscoelasticity after resting.     

Aeration of bread dough influenced by different way of processing

The effect of steady shearing versus z-blade mixing on mechanical aeration and gas retaining ability of the dough during processing and subsequent proofing and bread baking stages was investigated. Reduction in moisture content led to reduction in both static and dynamic densities of z-blade mixed dough. At low moisture content, dough had higher consistency and tended to physically entrap more air bubbles upon processing, leading to a higher dough volume and, thereby a low density. The results showed that both processes led to similar mechanical aeration as measured by static dough density immediately after processing. Shearing at a low rotational speed, led to similar proofing dough volume as z-blade mixing did. Nevertheless, both dough expansion test and breadmaking trials showed a significant reduction in gas retaining ability of sheared dough, especially at higher rotational speeds. This is explained by the fact that higher shear rates could break up the gluten network and negatively influence gas retaining ability. The results revealed the influence of processing conditions; e.g. the type of deformation flow on dough aeration. Furthermore, it was shown that rotational speed in the shearing system influences the aeration and gas holding ability of the dough during proofing and baking processes.     

Understanding the link between GMP and dough: from glutenin particles in flour towards developed dough

Clear correlations exist for glutenin macropolymer (GMP) quantity and rheological properties vs. wheat quality and dough rheological properties, but real insight in understanding these links is still missing. The observation that GMP consists of glutenin particles opens up new possibilities to reveal the underlying mechanism linking glutenin network properties with dough preparation. GMP was isolated from flour of three wheat varieties: Estica, Soissons and Baldus, strongly varying in their mixing requirements (expressed as time-to-peak, TTP). Decrease of GMP quantity and G′ vs. mixing energy was confirmed. More detail was obtained by studying the changes in GMP particles when mixing flour into dough. Mixing leads to a decrease in the average size of the particles. Interestingly, the TTP coincided with the work-input at which all particles just became soluble in SDS. At TTP, the average size of the GMP particles was the same for each variety. During mixing particles lost their globule shapes and appeared ruptured. Particle size analysis confirmed that particles were still present near TTP. Analysis of the change in particle size vs. energy input using physical principles revealed the following: (1) mixing energy is the predominant actuator in decreasing GMP particle size; (2) the initial GMP particle size in flour strongly determines the practical mixing requirements; and (3) the derived mixing energy vs. GMP particle size relationship was shown to be applicable for both Mixograph and Farinograph mixing. Our results demonstrate that, for the flour samples used, glutenin particle size determines TTP and GMP rheology, showing that glutenin particle properties could be a new key to understand the link between GMP and dough properties.     

Effects of vacuum mixing and mixing time on the processing quality of noodle dough with high oat flour content

The effects of different mixing parameters (vacuum mixing and mixing time) on oat (70% oat flour) and wheat noodle dough were investigated on the basis of textural properties and gluten formation. The results showed that at a vacuum degree of −0.06 MPa and mixing time of 10 min, oat and wheat dough sheets exhibited the highest resistance to extension and glutenin macropolymer (GMP) content, and had the most compact and uniform gluten network. Compared with wheat noodle dough, oat dough had lower resistance to extension, lower tightly bound water content, and higher GMP content. Microstructural examination showed that oat noodle dough had a more aggregated distribution of gluten protein compared with wheat noodle dough under the optimum mixing parameters. Furthermore, the poor binding ability of vital wheat gluten with water molecules caused the indexes of oat noodle dough to be more strongly affected by the changes in mixing parameters than wheat noodle dough.     

Mixing behaviour of a zero-developed dough compared to a flour–water mixture

The Z-blade mixing behaviour of zero-developed (ZD) doughs from the flours of two wheat cultivars of different gluten strength was compared to that of conventionally mixed dough made from the same flours. In farinograph experiments, use of ZD dough led to shorter development time (with less energy requirement), less stability time, and consequently earlier breakdown compared to conventional mixing of the corresponding flour–water mixture. Mixing of ZD doughs led to an almost similar decrease of glutenin macro-polymer (GMP) wet weight as that of doughs prepared from flour–water mixtures. However, comparison of wet weight of re-assembled GMP revealed that until time-to-peak (TTP) mixing, there was no difference in GMP recovery with respect to the starting material used in the z-blade mixing experiments. Beyond TTP, recovery of GMP in doughs prepared from both starting materials was reduced. The results of large-strain deformation rheology showed strong visco-elastic behaviour as characterised by the highest values of fracture properties (except ε_H), followed by a decline in those properties upon further mixing for doughs mixed from both flour–water mixture and ZD dough from both types of wheat cultivars. It was concluded that at mixing regimes before TTP, there was no difference between ZD doughs and flour–water mixtures in the mixer. When ZD dough is used as a starting material for dough preparation instead of flour, extra care should be taken not to over-mix the developing dough.     

The mesoscopic structure in wheat flour dough development

The Farinograph time-to-peak is an important wheat flour quality parameter. It is well-established that insoluble glutenins correlate with the strength of the gluten network and dough mixing time. To learn more about the physical changes at the mesoscopic level, dough samples were prepared in the Farinograph for study with diffusion wave spectroscopy. It was confirmed that a space-filling network was formed by wheat gluten proteins (mainly glutenin). At peak development (9.0 min) it was shown that the starch granules were confined in the gluten network. After the time-to-peak, dough resistance weakened, showing an increase of the starch granule movement. Kneading disrupts insoluble glutenin particles, the disrupted glutenin becomes part of the Sodium Dodecyl Sulfate (SDS)-extractable proteins. Both soluble and insoluble wheat protein extracts have been characterized by light scattering techniques. The results derived from light scattering of the wheat protein fractions: particle radii, apparent molar mass and geometrical shapes, suggests that the disrupted glutenin aggregate shape and glutenin size heterogeneity could be more important for gluten network bulk consistency, connectivity and resistance at dough peak, than the apparent molar mass of the solubilized glutenins, reaching a maximum after dough peak.     

Application of epifluorescence light microscopy (EFLM) to study the microstructure of wheat dough: a comparison with confocal scanning laser microscopy (CSLM) technique

To study dough microstructure, epifluorescence light microscopy (EFLM) combined with digital image processing software was used, which enabled an improved image quality. A comparison was made between EFLM and confocal scanning laser microscopy (CSLM) methods. Both techniques were satisfactorily able to demonstrate changes in the dough microstructure upon different stages of z-blade mixing. Dough mixed for a shorter time (under-mixed) showed a heterogeneous structure with coarse protein domains and clusters of starch due to local segregation or de-mixing effect. Increasing mixing time (optimal mixing) led to development of interconnected gluten network covering starch granules throughout the dough, representing optimal development. Over-mixing led to formation of a homogeneous dough microstructure in which the gluten phase showed a fine distribution throughout the dough. Using a double staining method in the preparation of samples for both microscopic techniques it was possible to observe gluten network structures together with starch granules. Moreover, special features of image processing software described in this study enabled us to improve EFLM images and to obtain comparable images with CSLM. This could favour a low cost and a convenient microscopic observation of biomaterials.     

Effect of Water Unextractable Solids on Gluten Formation and Properties: Mechanistic Considerations

A miniaturised set-up for gluten-starch separation was used to systematically study the effect of water unextractable solids (WUS) on the formation and properties of gluten. The results showed that WUS not only have a negative effect on gluten yield, but also affect gluten and glutenin macropolymer (GMP) composition and rheological properties. The negative effect of WUS on gluten yield could be compensated for to a large extent, but not completely, by increasing mixing time and mixing water. Adding xylanase can effectively counteract the effect of WUS. On the basis of these results we hypothesize that WUS interfere with gluten formation in both a direct and an indirect way. WUS interfere indirectly by competing for water and thus changing conditions for gluten development. This effect can be corrected for by the combination of adding more 0·2% NaCl solution during dough mixing and a longer mixing time. The particulate nature of WUS requires that the direct effect occurs through an interaction between WUS particles and gluten particles. Both effects of WUS can be counteracted through the use of xylanase.     

Correlation of glutenin macropolymer with viscoelastic properties during dough mixing

Although significant correlations exist for glutenin macropolymer (GMP) quantity and rheological properties/bread making quality of dough, little information about these links is available. The relationship between GMP contents measured by UV absorption method/RP-HPLC and dough viscoelastic properties determined by TA-XT2i texturometer from three wheat varieties (Xiaoyan6, Yumai56 and Zhengnong8805) during mixing was investigated. GMP contents of doughs decrease significantly (P<0.05) during mixing. During the initial mixing stage, amounts of the HMW-GS and LMW-GS and GMP decrease significantly (P<0.05). Their contents begin to increase beyond peak dough development time (DDT). This indicates that during further mixing after peak DDT some glutenin subunits are incorporated into GMP by repolymerization. The HMW/LMW-GS ratio has a significant effect on load-deformation properties (area, resistance and extensibility) of dough. The varieties behaved differently in relation to the contribution of their HMW-LMW-GS ratio to the rheological properties.     

How gluten properties are affected by pentosans

In the gluten-starch separation process gluten is formed first as a result of breakdown of the gliadin-glutelin structures during mixing followed by their re-agglomeration. To date the effect of pentosans and enzymes have not been studied separately. A simple modification of TNO Glutomatic system enables pentosans, enzymes, and other materials to be added after the mixing step allowing the effect of these additives to be studied separately. Using this technique, we observed that re-aggregation of gluten proteins starts immediately after the first mixing step during the dough dilution phase. Xylanase addition prior to dough mixing can lead to ‘overdose effects’ but these were not observed when xylanase was added later during the re-agglomeration phase. We were able to distinguish between physical and chemical effects of pentosans on gluten formation. The effect of water-extractable pentosans is only partly related to its viscosity, a ferulic acid (FA) related reaction is more important. Pentosans affect the affect the agglomeration by increasing the size of the glutenin macropolymer particles. When the water-extractable pentosan effect is prevented by xylanase or FA addition, aggregation during dilution is more extensive and the glutenin macropolymer has a lower average particle size with a resulting difference in gluten rheology.     

Evidence that pentosans and xylanase affect the re-agglomeration of the gluten network

In the gluten-starch separation process gluten is formed first as a result of breakdown of the gliadin-glutelin structures during mixing followed by their re-agglomeration. To date the effect of pentosans and enzymes have not been studied separately. A simple modification of TNO Glutomatic system enables pentosans, enzymes, and other materials to be added after the mixing step allowing the effect of these additives to be studied separately. Using this technique, we observed that re-aggregation of gluten proteins starts immediately after the first mixing step during the dough dilution phase. Xylanase addition prior to dough mixing can lead to ‘overdose effects’ but these were not observed when xylanase was added later during the re-agglomeration phase. We were able to distinguish between physical and chemical effects of pentosans on gluten formation. The effect of water-extractable pentosans is only partly related to its viscosity, a ferulic acid (FA) related reaction is more important. Pentosans affect the affect the agglomeration by increasing the size of the glutenin macropolymer particles. When the water-extractable pentosan effect is prevented by xylanase or FA addition, aggregation during dilution is more extensive and the glutenin macropolymer has a lower average particle size with a resulting difference in gluten rheology.     

Distribution and function of LMW glutenins,HMW glutenins,and gliadins in wheat doughs analyzed with ‘in situ’ detection and quantitative imaging techniques

The different gluten subunits, gliadins, LMW glutenins, and HMW glutenins have been reported to play different key roles in different type of wheat products. This paper studied the interaction between gliadin, LMW and HMW glutenins in soft, hard and durum semolina flour doughs during different stages of mixing. In order to see how do the gluten subunits (gliadin, LMW glutenin and HMW glutenin) redistribute during mixing, dough samples were taken at maximum strength and 10 min after maximum strength. The doughs have been mixed with the same level of added water (55%), therefore they all have different strengths values due to their changes in proteins content. Oscillatory rheological measurements were performed on the doughs. It has been found that HMW glutenins are relatively immobile because of their less molecular mobility and do no redistribute themselves especially at high strength for doughs such as hard wheat flour. LMW glutenins and gliadins on the other hand redistribute themselves at even at high dough strengths forming a more stable network. In weaker doughs such as soft wheat, the breakdown of the three proteins subunits is responsible for the decay in dough strength. We have also visualized how the greater amount of LMW glutenins in semolina is in constant interaction with HMW glutenins and gliadins allowing the dough to maintain a stable strength for an extended mixing time. Finally, we have found the ‘in situ’ detection and quantitative analysis techniques to be more sensitive to the changes occurring in the gluten network of the dough than the oscillatory rheological analysis.     

Phytate negatively influences wheat dough and bread characteristics by interfering with cross-linking of glutenin molecules

The influence of added phytate on dough properties and bread baking quality was studied to determine the role of phytate in the impaired functional properties of whole grain wheat flour for baking bread. Phytate addition to refined flour at a 1% level substantially increased mixograph mixing time, generally increased mixograph water absorption, and reduced the SDS-unextractable protein content of dough before and after fermentation as well as the loaf volume of bread. The added phytate also shifted unextractable glutenins toward a lower molecular weight form and increased the iron-chelating activity of dough. It appears that phytate negatively affects gluten development and loaf volume by chelating iron and/or binding glutenins, and consequently interfering with the oxidative cross-linking of glutenin molecules during dough mixing. Phytate could be at least partially responsible for the weak gluten network and decreased loaf volume of whole wheat flour bread as compared to refined flour bread.     

Rheological Properties of Dough During Mechanical Dough Development

During mechanical development dough is subjected to both shear and extensional deformations. Thus, it is expected that both flow conditions contribute to the development of dough. In order to monitor rheological changes, occurring during mixing, shear and extensional properties of dough prepared with two flours of different strength and various levels of mixing energy were determined using fundamental rheological methods. Rheological measurements included: small deformation and large deformation (shear test), planar extensional flow and a combined shear/extensional flow test, namely extrusion test. Results obtained in this research showed that, during mixing, dough develops with an increase in both apparent shear and extensional viscosities. For all the tests, plots of the measured rheological properties as a function of the mixing energy resembled typical mixing curves. This indicated that the increase in the power drawn by the mixer motor is due to the increase in both apparent shear and extensional viscosities. After peak dough development these properties decreased synchronously with the mixing curves. Results from small deformation shear tests exhibited large variability, particularly when non-mixed and underdeveloped doughs were tested. This variability was associated with poor water distribution in the sample due to insufficient mixing. Results of large deformation tests, including shear, planar extensional flow and the extrusion test, were less variable and showed that mixing and type of flour affect the rheological properties of dough.     

Influence of gliadin removal on strain hardening of hydrated wheat gluten during equibiaxial extensional deformation

The aim of the present work has been to study the equibiaxial extensional deformation of doughs of gluten- and glutenin-rich fractions containing 40 wt% water subjected to lubricated squeezing flow with four different crosshead speeds at room temperature. The gluten dough shows strain softening and hardening in succession whilst the dough where the gliadins have been removed by alcohol extraction does not show strain hardening behavior but breaks immediately after strain softening. The equibiaxial extensional viscosity decreases with increasing strain rate at given strains, appearing as strain rate thinning behavior, which is stronger in the glutenin dough than in the gluten dough. The large extensibility with strain hardening in the gluten dough is due to the presence of gliadins acting as both plasticizers and promoters for the more extensible networks.     

Functional Properties of LowMrWheat Proteins.III. Effects on Composition of the Glutenin Macropolymer During Dough Mixing and Resting

The effect of lowM_rwheat protein addition on the amount and composition of the glutenin macropolymer (GMP) of dough was investigated for the three wheat cultivars Obelisk (weak), Camp Remy (medium strong) and Rektor (strong). During mixing, the amounts of high and lowM_rglutenin subunit classes, and of the individual subunits decreased. The proportion of highM_rglutenin subunits decreased and that of lowM_rglutenin subunits increased, indicating an inhomogeneous distribution of the two subunit classes within the polymers present in GMP. During resting, the amounts of the glutenin subunit classes and of individual subunits increased. Meanwhile, the proportion of highM_rglutenin subunits in GMP increased. LowM_rwheat protein addition retarded re-polymerisation in that the amounts of glutenin subunit classes and of individual highM_rglutenin subunits in GMP increased less than without addition. The proportion of highM_rglutenin subunits in GMP directly after mixing was also decreased by lowM_rwheat protein addition, and the proportion increased faster during dough resting, compared with the GMP in dough without lowM_rwheat protein addition. Eventually, after 90 or 135 min resting, no differences existed in the proportions in GMP from doughs with and without lowM_rwheat protein addition. LowM_rwheat protein addition had no specific effect on individual highM_rglutenin subunits, nor on the x-type/y-type subunit ratio in the GMP. In contrast, with increasing lowM_rwheat protein addition, a highly significant reduction in the subunit 10 or 12/subunit 9 ratio in GMP was observed. This finding is in line with the decrease in this ratio directly after mixing in GMP of the dough without lowM_rwheat protein addition. Since no specific effects were observed, it can be concluded that the lowM_rwheat protein acts rather unspecifically on the GMP of dough.     

Hydration,polymerization and rheological properties of frozen gluten-water dough as influenced by thermostable ice structuring protein extract from Chinese privet (Ligustrum vulgare) leaves

The effects of thermostable ice structuring proteins (TSISPs) extracted from Chinese privet (Ligustrum vulgare) leaves on water molecular state, dehydration of gluten proteins, secondary structure of proteins, glutenin subunit of glutenin macropolymer (GMP) and rheological properties of gluten doughs during frozen storage were investigated by nuclear magnetic resonance (NMR), attenuated total reflectance-Fourier transform infrared reflectance (ATR-FTIR), reversed phase-high performance liquid chromatography (RP-HPLC) and dynamic rheometry. After frozen storage for 5 weeks, the control sample showed dehydration of gluten proteins and mobility of water molecules in gluten dough increased, significantly indicating ice formation and water redistribution. Secondary structure of gluten proteins changed significantly, α-helix decreased and β-sheet increased. Glutenin subunits depolymerized, indicated by the decrease in high molecular weight glutenins/low molecular weight-glutenins (HMW/LMW) ratio. The decrease in elastic moduli (G′) and viscous moduli (G′') showed the deterioration of rheological properties of gluten dough. The addition of TSISPs inhibited the dehydration of gluten proteins, decrease in α-helix, increase in β-sheet and HMW/LMW ratio, resulting in improved rheological properties of gluten dough.     

Rheological Behaviour of Wheat Glutens at Small and Large Deformations. Effect of Gluten Composition

Glutens derived from two wheat cultivars with a known difference in bread making quality, i.e. cv. Katepwa (good) and cv. Obelisk (poor), were fractionated into gliadin and glutenin. Cultivar Katepwa gluten contained more glutenin than cv. Obelisk gluten. Reconstituted glutens were prepared by mixing, in different ratios, gliadin and glutenin fractions that originated from one gluten type or from both glutens. The rheological properties of these mixtures, when hydrated, were studied at small deformations in shear and at large deformations in biaxial extension. The reconstitution of gluten in its original glutenin/gliadin ratio produced a composite that had a somewhat higher resistance to deformation and was more elastic than the unfractionated gluten. This was true for both gluten types. However, the difference between the rheological behaviour of both reconstituted gluten types was comparable with that found between the native glutens. From measurements with glutens reconstituted at various glutenin/gliadin ratios, it appeared that the main factor determining the rheological behaviour of hydrated gluten is the glutenin/gliadin ratio. By interchanging the gliadin and glutenin fractions of the two glutens, it was shown that the source from which the fractions originated, particularly that of the glutenin fraction, was also important.     

Mixing history affects gluten protein recovery, purity, and glutenin re-assembly capacity from optimally developed flour–water batters

Batters, from three wheat cultivars, were mixed up to their maximal consistency (t_peak) at different mixing speeds (N) and flour/water ratios [Auger, F., Morel, M.H., Lefebvre, J., Dewilde, M., Redl, A., 2008. A parametric and microstructural study of the formation of gluten network in mixed flour–water batter. Journal of Cereal Science 48, 349–358]. Gluten and starch were extracted from those batters using a process which included two successive steps: dilution and sieving. In order to reveal the specific influence of the mixing step, a standardized gentle washing and sieving procedure was selected. Mixing the batters at t_peak guaranteed a high and stable gluten protein recovery (ca. 82%) irrespective of mixing conditions. SE-HPLC analysis of protein, from flours and batters sampled at t_peak, demonstrated that mixing led to the almost total breakdown of the unextractable glutenin polymers (ca. 80%), whereas their re-assembly occurred during gluten extraction. The extent of glutenin re-assembly in gluten was influenced by the batter mixing history and was mainly related to the number of mixing rotations (N.t_peak). Gluten protein contents were also found related to N.t_peak. We proposed that the leaching of starch from the batter during gluten extraction was controlled by the elasticity of the protein network, i.e. the gluten content in unextractable glutenin. An innovating scheme relating the glutenin re-assembly capacity to the irreversible thiol protein oxidation is proposed.