The RVA pasting curve of one of the soybean flours is shown in Figure 6.1. Significant differences were observed in the pasting characteristics of flours from different soybean cultivars and especially in the RVA peak viscosity (Table 6.4).
0 50 100 150 200 250 300 350 400 450 500 0 2 4 6 8 10 12 14 16 18 20 Time [min] V isco si ty [ R V U ] 0 10 20 30 40 50 60 70 80 90 100 T e m p er at u re [ °C ] Viscosity [RVU] Temp Peak viscosity Peak time Slope start
The details of thermal gelation vary with the kind of protein used, and reflect the unique primary structure of the particular protein and the kinds of bonds which contribute to the secondary and tertiary structure of the proteins. The RVA, a cooking viscosimeter, is useful for simulating the effects of thermal processing and changes in environmental conditions on the rheological properties of food proteins. A mixture of soybean flour in water represents a complex system consisting of protein, polysaccharide and lipid. While protein is the main component of soybean flour, polysaccharides (including a small amount of starch) may substantially affect the gelling properties of such a system due to their high water absorbing capacity. Furthermore, soybeans contain around 20% oil which, under high temperature, is likely to interact with polysaccharides and possibly form complexes. According to Zhang and Hamaker (2003), the production of the cooling stage viscosity peak in the RVA profile indicates a three way interaction. Although the RVA curves of most soybean varieties produced the cooling stage viscosity peak, we decided not to include this parameter in the results. As shown in a previous study (Blazek, 2005), the repeatability of RVA tests was satisfactory only in the first half of the test. The relatively smooth traces became jagged in the second half of the test leading to considerable variations in viscosity values at the end of the test when the soybean gel was cooled down.
Table 6.4: RVA pasting characteristics of flours from different soybean cultivars
Variable Minimum Maximum Mean
RVA visc. [RVU] 184 457 306
RVA peak time [s] 468 835 630
RVA slope start [s] 267 302 282
3-g RVA visc. [RVU] 212 445 347
3-g RVA peak time [s] 495 745 625
When environmental conditions favourable to gelling are created, a progressive cross-linking mechanism takes place. In the RVA canister, increasing clusters of associated
chains develop until a critical point is reached where the network spans the whole sample volume. The gel point and the network formation are accompanied by a sharp increase in viscosity. The network continues to further increase in elasticity and stiffness until a quasi- stable state is attained, provided there are no interfering effects such as syneresis.
Preliminary studies (Turner et al., 1996; Blazek, 2005) show the potential of the RVA to detect quality differences in proteins. Tests with sample weights adjusted to contain a constant 3.0 g protein level showed substantial differences between RVA viscosity curves. This indicates that the viscosity results were affected by factors additional to simple protein content. The significance of these results is discussed later.
When a constant amount of soybean flour (8.0 g) is used, it’s likely that the quality differences in protein are suppressed by the differences in protein content. This is supported by the strong positive correlation between the RVA viscosity and the soybean protein content (r = 0.732, P < 0.001; Figure 6.2). A positive correlation was also found between the WAC and RVA peak viscosity (r = 0.536; P < 0.01) and between WAC and RVA slope start (r = 0.521; P < 0.05).
0 100 200 300 400 500 32.0 33.0 34.0 35.0 36.0 37.0 38.0 39.0
Soybean protein content [%]
R V A pe ak v is cos ity [R V U ]
Figure 6.2: The correlation between the RVA peak viscosity and the soybean protein content (r = 0.732, P < 0.001).
6.3.4 Protein subunit composition
If 11S and 7S can be distinguished with SDS-PAGE analysis of soy protein extracts, the relative content of 11S and 7S, as well as the subunits can then be determined. As an example, a SDS electrophoretogram of one of the samples is shown in Fig. 6.3. The area and intensity of the peaks varied substantially among cultivars. The method of Liu et al. (2007) was used where the SDS-PAGE patterns of the soybean protein extracts were divided into two regions: the region of peaks with MW < 44 kDa and that with MW > 44 kDa. The first region containing mainly 11S proteins was divided into four parts, called subunit groups, i.e. 11S-1 (14.4–22 KDa), 11S-2 (22–26 KDa), 11S-3 (26–34 KDa) and 11S-4 (34–44 KDa). The second region containing mainly 7S protein was divided into six subunit groups, i.e. 7S-1 (44–49 KDa), 7S-2 (49–55 KDa), 7S-3 (55–67 KDa), 7S-4 (67– 73 KDa), 7S-5 (73–82 KDa) and 7S-6 (82–91 KDa). Integrating the area of peaks within a certain range of molecular weights was used to calculate the relative content of each protein subunit. The sum of relative contents of 11S-1 to 11S-4 was obtained as the relative
content of 11S protein, those of 7S-1 – 7S-6 as that of 7S protein, and therefore, the 11S/7S ratio obtained. The proposed criteria were demonstrated to be simple, stable and feasible.
Figure 6.3: The electrophoreogram of the protein extracted from one of the varieties used in the study (myosin and bradykinin used as the upper and lower internal marker).
The range of clear peaks per cultivar was between 10 and 15, with a mean value of 12 within MW 14–220 KDa (the range of standards of protein MW markers). None of the samples showed any peaks within the range of subunit 7S-4 and an insignificant average amount of protein fitted within the range of subunit 7S-1, with the highest average relative content of 41.9% corresponding to the subunit 11S-4.
The relative average content of protein subunit groups averaged over the 20 soybean samples is shown in Table 6.5. The 11S relative content varied between 63.1 and 81.3% with an average of 72.1%, 7S relative content varied between 17.8 and 36.8% with
an average of 27.7%, and 11S/7S ratio varied between 1.71 and 4.57 with an average of 2.70. The results indicated a great diversity in protein subunit composition.
Table 6.5: The relative average content of protein subunit groups in 20 protein extracts
11S protein 7S protein
Sub-group MW Percent Sub-group MW Percent
(kDa) of total (kDa) of total
11S-1 14.4 – 22 4.2 7S-1 44 – 49 0.1 11S-2 22 – 26 25.0 7S-2 49 – 55 4.8 11S-3 26 – 34 1.0 7S-3 55 – 67 0.6 11S-4 34 – 44 41.9 7S-4 67 – 73 0.0 7S-5 73 – 82 10.8 7S-6 82 – 91 11.5
Average distribution of bands with various molecular weights from 14.4 to 91.0 kDa.
In each cultivar, the sum of relative content of all subunits in MW 14.4 – 44 kDa and in MW 44 – 91 kDa was regarded as the relative content of 11S and 7S components of a cultivar, respectively. The average relative content means the relative protein contents of all electrophoretic ranges with a same molecular weight range averaged over the 20 soybean samples.
Principle component analysis was performed to determine which variables correlate with 11S/7S ratio and could therefore indicate the quality of soybean protein. A positive correlation was observed between 11S/7S ratio and the 3-g RVA viscosity (r = 0.485, P < 0.05). Soybean ash content strongly correlated with the 11S/7S ratio (r = 0.639, P < 0.001). Interestingly, a negative correlation was found between 11S/7S ratio and WAI (r = -0.501, P < 0.05) which, however, seems to be in agreement with Bresnahan et al. (1981) who observed an inverse relationship between 11S protein level and water absorption when studying the texture formation.