1 Value Added Wheat CRC, North Ryde, NSW, 1670, Australia
2 Allied Mills Ltd, Summer Hill, NSW, 2130, Australia
3 University of Sydney, Camperdown, NSW, 2007, Australia
4 Food Science Australia, North Ryde, NSW, 1670, Australia
Sedimentation testing techniques are used widely in the United States, Europe and the United Kingdom as a means of measuring gluten strength (Carter, et al. 1999). These sedimentation techniques involve hydration of a small flour sample in a lactic acid solution containing either the detergent sodium dodecyl sulphate (SDS sedimentation) or isopropyl alcohol (Zeleny sedimentation). The presence of lactic acid in solution causes the hydrated flour particles to sink in the form of a sediment, the level of which, indicates the strength of the gluten. High sedimentation volume indicates strong gluten while low sedimentation volume indicates weaker gluten.
Despite the longevity, relative economy and simplicity of these tests, relatively little is known about how the sediment forms or the reasons for differentiation between samples in terms of G x E interactions. Adayemi and Müller (1983) speculated that lactic acid ruptures the walls of the endosperm cells causing hydration of the endosperm particles and that this causes formation of proteinaceous ‘fibril’ molecules which bind the flour particles together. Further, they considered that these fibril structures were stabilised by disulphide linkages. This concurs with an earlier study by Orth et al. (1973) which indicates that addition of disulphide cleaving agents to the SDS/lactic acid solution retards sediment formation.
This study aims to observe the effects of a number of bond cleaving reagents on sedimentation test results and also to observe the influence of certain genetic and environmental factors.
Materials and methods
Sample acquisition and preparation
All samples were grown in the northern New South Wales/southern Queensland area and were acquired through Allied Mills Toowoomba. Clean grain samples were either ground to meal on a Perten Falling Number Mill or conditioned and milled on a Bühler Laboratory Mill.
Sedimentation testing of all samples was carried out at Allied Mills Summer Hill using an automated mixing rack in accordance with a modified version of AACC Method 56-70.
Protein bond cleavage
Bond cleaving agents were made up as detailed below:
1) Disulphide bonds - 0.01M and 0.1M L-Cysteine,
2) Ionic bonds - 0.1M and 1M ammonium chloride
3) Hydrogen bonds - 0.5M and 1M urea,
50mL of these solutions was added to each measuring cylinder, prior to wheat/meal being added, in place of the 50ml of water usually used. Otherwise sedimentation procedure was as above.
Artificial sprouting of grain
A 100g subsample of each grain sample was taken and these samples were soaked in water for one hour before being drained and patted dry. These samples were then spread out on damp paper towels in a shallow tray, covered with plastic wrap and left at 25ºC for 24 hours. Following this, both the plastic wrap and paper towels were removed and the samples dried for 24 hours at 35ºC. Falling number evaluations were carried out for sound and sprouted grain using AACC Method 56-81B without correcting values for 14% moisture content.
These values were then used with the scale of Perten Liquefaction Number to calculate the proportion of sprouted grain required for blending with sound grain to achieve falling number values of 100 and 200. The meals were then blended in the appropriate ratios and again tested for falling number and sedimentation volume.
Environmental conditions and data acquisition
Grain and flour sample pairs grown in the 2003-04 harvest were used for this work. Wholemeal samples underwent the following tests at Allied Mills, Summer Hill: SDS-
sedimentation, NIR (AACC Method 39-25). Flour samples also underwent NIR testing as well as Zeleny sedimentation (AACC Method 56-60), Extensograph (RACI Method 06-01) and Farinograph (RACI Method 06-02). Environmental data was obtained from the Bureau of Meteorology.
Results and discussion
Protein bond cleavage
Addition of L-cysteine to the vessel had a severe impact on the resulting sediment volume at the conclusion of the test (Figure 1). This was evident for both flour and wholemeal samples though deterioration was more severe for flour samples at both high and low concentration.
Figure 1: Effect of L-cysteine on SV for 1a) wheatmeal and 1b) flour.
Sedimentation volume (SV) also decreased drastically in the presence of both high and low concentrations of ammonium chloride. Again, flour samples were more severely affected though the wheatmeal samples responded much differently low concentration. Sunbrook, Sunstate and Qual2000 all experienced decreases in SV of about 10% while Sunco remained unchanges and the SV for Rosella actually increased approximately 10% (Figure 2).
A similar, more pronounced effect was seen with the addition of urea (Figure 3). In this case significant increases in SV were observed for most of the wholemeals and Sunbrook and Rosella remaining unchanged. Mild decreases were observed for all flour samples with the exception of Rosella which again remained unchanged.
Figure 2: The effect of ammonium chloride on SV for 2a) wheatmeal and 2b) flour
Figure 3: The effect of urea on SV for 3a) wheatmeal and 3b) flour
These results indicate that cleaving either ionic or disulphide linkages within a protein structure, results in severe damage to the functional ability of the protein in terms of flocculation and sediment formation. Cleavage of hydrogen bonds however appears to result in a reduced, though still significant overall loss of protein function.
Effects of rainfall and temperature
Table 1 details the manner in which standard quality tests for flour and wholemeal were affected by environmental conditions in terms of correlation coefficient. The strongest correlations were observed for Zeleny sedimentation of flour. These results indicated that Zeleny sedimentation (both as SV and protein quality value (PQV)) is strongly affected by the environmental conditions to which the grain is exposed prior to harvest. Positive correlations with rainfall existed for protein and moisture content as determined by NIR (NIRP and NIRM respectively), SDS SV, dough stability and resistance to extension while dough stability and resistance to extension also displayed negative correlations with days over 35ºC.
Table 1: Correlations of quality test results with number of days over 35ºC and rainfall in the six weeks prior to harvest.
Figure 4 illustrates the changes in sedimentation volume as a result of changes in falling number value of wheatmeal. The three flours chosen for this study were selected to represent a range of sedimentation behaviours with Rosella performing poorly, Sunstate achieving average results and Sunco performing very strongly.
Figure 4. Effect of sprout damage on sedimentation volume for weak, medium and strong flours
These results indicate that as the level of sprout damages increases, sedimentation volume increases as well. This is particularly obvious with the variety Sunstate for which a linear relationship exists between decreasing falling number and increasing SV. This trend can also be observed for Rosella however, the rise in sedimentation volume occurs only when the grain is heavily sprouted. This may be due to the original falling number of the weaker variety being relatively low compared with the other varieties. Sunco on the other hand, showed a slight increase with low levels of sprouting however the result decreased again with heavy sprouting.
It can be concluded from this study that both disulphide linkages and ionic bonding play a crucial role in floc formation during sedimentation testing. When these bonds are cleaved or prevented from forming the resulting sediment is looser and heavier resulting in much lower sedimentation volumes.
Further, current sedimentation testing is likely to be inappropriate as a means of testing starch damaged grain as sedimentation results increase with decreasing falling number. This is likely to result in inaccurate prediction of baking quality. Zeleny sedimentation however, proved to be a useful indicator of the effects of growing environment.
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