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Size distribution of polymeric protein in soy-wheat doughs during fermentation

E. Maforimbo1, G. Skurray1, S. Uthayakumaran2 and C.W. Wrigley2

1University of Western Sydney, Hawkesbury Campus NSW 1797, Australia
2Food Science Australia and Wheat CRC, North Ryde NSW 2113, Australia


The present paper explores the hypothesis that a process of physical modification of soy flour (moist heat treatment) causes an increase in the molecular-weight distribution of the soy proteins, thus making them more suitable for dough formation. This treatment has been reported to produce a 1:1 soy-wheat dough that exhibits higher resistance to extension (Rmax), greater tolerance to mixing, better mixing stability, higher water uptake and better water absorption than a 1:1 soy-wheat composite dough made from raw soy flour (RSF) (Maforimbo et al. 2005). Although rheological properties indicated that interaction of components could be better in soy-wheat dough made from physically modified soy flour (PMSF), it was found necessary to evaluate protein interactions in the soy-wheat doughs. Size-exclusion high-performance liquid chromatography (SE-HPLC) was used to determine the protein compositions of these composite doughs during fermentation, thus to gain a better understanding of protein size distribution and possible interactions at the molecular level. Molecular size distribution of proteins is an important indicator for determining dough strength and thus baking quality; an indication of size distribution is provided by determining the proportion of large glutenin polymers as “unextractable polymeric protein” (%UPP) (MacRitchie, 1992). Capillary electrophoresis (Lab-on-a-chip) was also carried out to determine if protein interactions involved disulphide covalent bonds or not.

Materials and methods

Preparation of soy flours

Whole-seed soybeans (Meriram Pty Ltd, Everton Hills, NSW, Australia) were use to produce physically-modified soy flour (PMSF). Soybeans were dehulled (mechanically) and flush-steamed for 3 minutes at atmospheric pressure to deactivate enzymes. The beans were spread on stainless-steel trays and blow-dried with hot air (80C) to constant weight in an oven for 3 hours. The cooled beans were later milled to fine flour through a 0.8 mm sieve, using a hammer mill (Newport Scientific Cereal Mill 6000 model, Warriewood, NSW, Australia). The raw soy flour (RSF) was full fat, enzyme-active whole soy flour (Meriram Pty Ltd), made by milling the raw soy beans as above. Commercial strong-wheat baking flour was obtained from Centerion Milling Company, Pty Ltd. Melbourne. All flours were stored in a cold room at 4C. The moisture content for these flours were 6.0, 5.8 and 11.57 g/100 g, and the protein content (as is basis) was 33.8, 36.8 and 12.5 g/100 g for RSF, PMSF and the wheat flour, respectively.

Rheological tests

For Farinograph and Extensograph testing, the soy-wheat doughs were prepared by mixing wheat and soy flours in a 1:1 ratio, using either of the two soy-flour types, physically-modified soy flour (PMSF) and raw soy flour (RSF). Farinograph analysis was performed following methods from Preston & Kilborn (1984), using the Do-Corder Brabender OHG (Duisburg, Germany). The amount of water used for the dough was the optimum water for Farinograph absorption at 500 Brabender units (BU). Extensibility (Ext) and maximum resistance (Rmax) were determined by the Extensograph method of Rasper (1991) using the Brabender Duisburg mod Exek/7, Nr.779, (Germany).

Determination of UPP% (SE-HPLC analysis)

Dough mixes (approximately 2 grams) from the above experiments were allowed to ferment in an oven (without yeast); they were later freeze-dried and ground to provide uniform powders. Protein size distribution was determined by SE-HPLC, using the modified method of Batey et al. (1991). A running time of 10 min was used (flow rate 2 mL/min) instead of the standard 35 min run (0.5 mL/min). The eluent used was aqueous acetonitrile buffer (0.05% trifluoroacetic acid in water and 0.05% in acetonitrile). The proteins were detected at a wavelength of 214 nm.

Lab-on-a-chip Capillary electrophoresis

Capillary electrophoresis, (Lab-on-a-chip) was determined on the dough powder samples following methods from Uthayakumaran et al. (2004). Dough mixtures from above were heated in a fan oven for 3 mins at 90C, after which they were freeze dried under nitrogen and ground to powder before analysis. N-ethylmaleimide (NEMI) was used to block SH groups in control doughs during heating (Roesc & Corredig, 2005). The amount of NEMI used was the equivalence of free SH groups in the dough mixtures.

Results and discussions

Rheological parameters and protein size distribution (UPP %) for soy-wheat doughs

The rheological parameters (Table 1) show that there were considerable decreases in maximum resistance to extension (Rmax) and stability in the W-RSF (wheat – raw soy flour) dough, compared to the wheat dough. The arrival time of the dough to touch the 500 BU line has been slowed down considerably in the W-RSF dough. On the other hand, the wheat-PMSF dough had better stability and Rmax, although its extensibility (E) was still well below that of the wheat dough. The wheat-PMSF dough had a greater mixing tolerance than the W-RSF dough, although it was well short of the value for wheat dough.

Table1. Comparison of rheological properties and unextractable polymeric protein (UPP) results for wheat dough and soy-wheat dough at 1:1 ratio of soy and wheat flour. Values shown are the means of duplicate analyses; error was 2% of the mean.


Arrival time/min


aRmax bBU 45 min

cE /cm at 45 min

%UPP at0 min

%UPP at1 hour

%UPP at2 hours

Wheat dough
























a Rmax = maximum resistance to extension of the dough b BU = Brabender Units
E = dough extensibility

The proportion of large protein polymers (as %UPP) was determined for the soy-wheat doughs (Table 1) at three points during fermentation (non-yeasted) in an oven at 37C. The percentage of unextractable polymeric proteins was expectedly highest in the wheat dough. The %UPP was drastically reduced in W-RSF dough, while the W-PMSF dough had a much higher level of polymeric protein than for the unmodified soy, although it was still short of the value for the wheat dough. These differences in %UPP results, also reflected in the rheological results, are postulated to explain the improved dough-making properties of PMSF compared to RSF.

Figure 1a

Figure 1b

Figure 1c

Figure 1. Lab-on-a-chip capillary electrophoresis for soy-wheat dough extracts, shown 1a, as simulated gel patterns and 1b, as elution profiles (non-reducing conditions with 1% SDS). Figure 1a: Protein extracts from heated doughs; lane 1, ladder of protein standards, showing their sizes in kilo Daltons; lane 2, wheat dough; lane 3, NEMI added to wheat dough; lane 4, W-PMSF dough; lane 5, NEMI added to W-PMSF dough; lane 6, PMSF dough. Figures 1b and 1c show the elution profiles corresponding to lanes 2/3 and lanes 4/5; profiles in bold represent dough without NEMI, while the thin lines represent the NEMI-treated doughs.

Capillary electrophoresis

Heat and NEMI treatments were applied to the dough samples in an attempt to identify any disulphide-bond interactions as a result of heat treatment (Figure 1). Electrophoregrams for extracts of wheat and W-PMSF doughs had similar elution profiles in terms of protein size distribution (Figure 1b and 1c). However, the presence of large protein aggregates was not evident in these capillary electrophoregrams; apparently this form of capillary electrophoresis does not show the presence of the very large aggregates seen in SE-HPLC profiles and estimated as UPP (Table 1). Instead, there was a first large peak (corresponding to a molecular weight (MW) of <20 kDa) and a second peak of modest size (MW of 50-60 kDa).

Heat treatment (90C for 3 minutes) was applied to these doughs, in the presence and absence of NEMI, which acts as a blocker of disulphide-bond interactions. There were only small differences in profile as a result (Figures 1b and 1c). It can thus be concluded that disulphide-bond interactions are minimal at this stage of the baking process due to the level of heat applied. Further experiments of a similar nature may help to determine whether such interactions are involved during the physical modification treatment when raw soy is physically modified by heat treatment. This possibility seems likely, because analyses of the SS and SH groups of raw and of modified soy showed a large increase in the ratio SS:SH as a result of the initial heat treatment of physical modification.


These experiments indicate that soy-wheat composite flours, using physically modified soy, produce stronger doughs with potentially better baking qualities than those made from raw soy, because the modification process causes an increase in the size distribution of the soy proteins. Specific details of the increased size distribution were provided by size-exclusion high-performance liquid chromatography. Capillary electrophoresis (using Lab-on-a-chip methodology) was less useful as a means of examining the size distribution, as it did not reveal the presence of very large proteins, which are presumed to be critical to the improvements provided by the physical modification process. This heat treatment of raw soy is simple, appearing to be suitable for use in village situations in Zimbabwe as a means of improving the dough and baking qualities of soy-wheat composite flours.


The support from the University of Western Sydney in sponsoring this work is greatly acknowledged. Special thanks are extended to CSIRO for facilitating the analysis and expertise for this work.


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Maforimbo, E., Nguyen, M., and Skurray, G. R. (2005). J. Food Engineering (in press).

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Uthayakumaran, S., Batey, I.L., and Wrigley, C.W. (2005). J. Cereal Sci. 41: 371-374.

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