Сельскохозяйственная биология, 2010, № 3, с. 63-70.

УДК 633.11:631.524.7:57.08

ESTIMATION OF GRAIN QUALITY IN SOFT WHEAT VARIANTS BY SDS-SEDIMENTATION

V.P. Netsvetaev^1,2, O.V. Lyutenko¹, L.S. Pashchenko¹, I.I. Popkova¹

On the variants of a winter soft wheat harvest of 2007-2008 years the authors compare the results of estimation of grain quality on gluten wash and sedimentation by standard and own modified methods. The presented techniques permit to isolate the forms with extra-high quality of grain gluten in collection and hybrid material.

Key words: IDK-analysis, gluten, Triticum aestivum, sedimentation tests, disulfide bonds.

Estimation of grain quality by SDS-sedimentation is the extensively used method in studies of durum wheat (1-3). For a soft wheat, the sedimentation in dilute solutions of organic acids - Zeleny test and its modification of Pumpyansky-Sozinov (4-6) – are applied more often, though, in recent years, SDS-sedimentation is increasingly being introduced in studies of a soft wheat.

The purpose of our experiments was to estimate grain quality in the selected grain samples of soft wheat using classic and modified SDS-sedimentation tests.

Methods. The objects of our study were the samples of winter soft wheat obtained by competitive and preliminary tests  harvested in 2007 (74 forms) and 2008 (68 forms), including the locally adapted varieties and  the varieties in process of state tests. For IDC-tests (measuring the gluten deformation index on IDC-1 device), the grain was milled to the flour meal using the mill ML-1 (type Cyclone, Russia), the flour for sedimentation was obtained on the roller mill Quadrumat® Junior («Brabender», Germany) according to recommendations (6).

SDS-sedimentation tests were performed in two versions. The first version (SDS 1) was based upon the methods of A.I. Rubalka (2001, personal report). The selected sample of flour (3.2 g) was placed in a graduated cylinder volume 100 ml and 50 ml 2% acetic acid was added, then stirred by rocking for 30 seconds and left to precipitate for 5 min at room temperature. Then 50 ml of the solution containing 100 g SDS in 5 liters of 2% acetic acid was added. The mixture was stirred by rocking for 10 seconds, precipitated for 5 minutes and the first account of swelling capacity was performed (ml). The procedure was repeated (stirred for 10 s, precipitated for 5 min), after that the second account was done  and the average value was calculated.

In the second version (SDS 2), to assess the swelling capacity (sedimentation) after the destruction of disulfide bonds in protein aggregates,  the sample of flour (6 g) was placed in measuring cylinder volume of 100 ml, 1% solution of sodium sulphite (Na₂SO₃) was added, then stirred for 30 seconds and left to precipitate for 30 min, and after that 50 ml of the solution containing 100 g SDS in 5 liters of 4% acetic acid was added. The mixture was stirred by rocking for 10 seconds, precipitated for 5 minutes, and the rate of sedimentation was measured. The procedure was repeated and the average value was determined.

The sedimentation method of Pumpyansky-Sozinov (macromethod; 2% acetic acid solution, 3,2 g flour sample, measuring cylinder volume of 100 ml) was performed as described (6).

The standard definition of gluten quality with washing off from a flour meal was carried out under the prescribing of A.A. Sozinov et al. (6). According the method modification of V.P. Netsvetaev et al. (7, 8), the flour meal was soaked in the acetic acid solution (0.05 M or 0.20 M - the variant I and II, respectively): a dough was made in a porcelain mortar using a spatula at a temperature of 18 ± 2 °C , shaped into a dough ball, then placed in a mortar or cup, covered with lid and left for 20 minutes; then gluten was washed in a gentle stream of water at the same temperature over a flour sieve (silk № 38  or capron № 49). The removing pieces of gluten were collected from the sieve and combined into the overall sample. To remove residual water, the gluten was pressed in a handful until the state when it became sticky in hand, then weighed up to 0,1 g; after that gluten was washed for 2-3 minutes, wringed and weighed again. When the difference between the weight values did not exceed ± 0,1 g, washing was considered complete. After assessing the quantity and quality of wet gluten, it was put on a piece of paper and placed into a desiccator oven for 4 hours at 105 °C.  The amount of wet and dry gluten was expressed as a percentage to a flour sample weight.

The quality of wet gluten was determined on the device IDC-1 (Russia) according manual; the device was calibrated before operating. 4 g sample of the washed and weighted gluten was shaped into a ball by pressing down 3-4 times, placed in a cup with water at 18 ± 2 °C for 15 minutes. Then the sample was put on the device sampling stage and exposed to the deforming load within 30 seconds.

The variance analysis of data was performed using StatNov program (9), and  correlation analysis and definition of the other statistical indicators – by the program Statistica 6.0.

Results. This is a well known fact (10-13), that technological properties of flour (grain) are determined by protein quality, which is primarily attributable to varietal characteristics of caryopsis storage proteins. Therefore, flour quality depends on hereditary variants of protein molecules in weevil endosperm. Physical and chemical properties of a dough are connected with a structure of protein molecules and their ability to form intermolecular aggregates (largely - due to intermolecular disulfide bonds stimulated by medium acidity). Different varieties are distinct in number and nature of these bonds (intrachain, interchain) in protein complexes of weevil endosperm. The presence of reducing agents (β-mercaptoethanol, dithiotriethol or sodium sulfite (Na2SO3) etc.) promotes disaggregating due to the destruction of intermolecular disulfide bonds. An alkaline environment promotes intensification of the process.

When Zeleny test and in SDS 1 version, flour swelling occurs in an acidic medium stimulating polypeptides aggregation. This fact was confirmed by comparing the results of standard and modified (I version) methods for determining gluten quality (Table 1). Since, for example, the defeat by grain chinch leads to gluten dilution and, accordingly, to increase in IDC, it is advisable to estimate flour physical properties using the authors’ own modified method of washing gluten, which allows to reduce the effect of non-hereditary factors including the effects of chinch enzymes.

Medium acidity (see Table 1.) contributed to a significant strengthening (IDC) and reduction of moisture content in gluten. Note: the studied samples were unequally defeated by grain chinch (0,1-3,0%, the average for the experiment - 1,2% grains), which resulted in obtaining the higher values (average - 96,1 ± 0,8 IDC units) at standard assessment than using the modified method (average - 80,8 ± 1,2 IDC unics), which indicates gluten dilution under the action of pest proteases. The more detailed analysis of gluten quality in wheat defeated by grain chinch was presented earlier (7, 8). Correlation analysis has revealed a significant correlation between sedimentation rates obtained by the method of Pumpyansky-Sozinov and SDS 1 method. Moreover, IDC values measured by the standard method were not correlated with the results found  in versions of Pumpyansky-Sozinov and  SDS 1, while the modified method showed the correlation (Table 2).

Therefore, medium acidity for sedimentation negate the changes of flour swelling caused by chinch defeat (see Table. 2, 3). No relationship was found between the level of pest defeat and grain quality indicators in versions of Pumpyansky-Sozinov and SDS 1, as well as using the modified method. Consequently, for estimation the hereditary properties of grain, it is appropriate to apply acidic medium conditions due to the reduced influence of environmental variability on test results.

Adding to a flour meal of reducing agents should lead to destruction of gluten complex. Indeed, in the variant with sulfite, gluten could not be wash off even in acidic conditions – it became a sticky mass. Knowing this effect of reducing agents on protein complex, the authors compared flour swelling properties determined by SDS 1 and SDS 2 methods. The difference in these rates, apparently, characterizes the number of intermolecular disulfide bonds in gluten complex.

In the first stage of SDS 2 processing (half-hour incubation of flour sample in a solution of sodium sulfite), the samples’ differentiation by swelling was observed. A small negative correlation of swelling at intermediate processing stage with the level of grain destruction by chinch was revealed (r =  0,31 ± 0,17; n = 34, 2008). Consequently, in an aqueous solution of sodium sulphite, proteolytic enzymes of grain chinch are active, able to destroy peptide bonds and may be responsible for about 10% variation of this index. In the second stage (final estimation), such correlation was not found (see Table. 3). At the same time, the significant positive correlation between SDS 2 index and the amount of wet and dry gluten. Characteristically, that SDS 2-method has led to the results different from those obtained by other sedimentation methods (see Table. 3), which indicates that SDS 2 depends on gluten content in grain (raising the number of protein molecules should increase swelling as the result of aggregation independent from disulfide bonds). Differentiation by sediment amount was insignificantly correlated with gluten physical properties (see Table. 3), thus confirming the role of intermolecular disulfide bonds in formation of gluten complex. In general, the experiment findings suggest that the amount of sediment measured by other sedimentation methods (Zeleny, Pumpyansky-Sozinov, SDS 1) is primarily provided by proteins containing cysteine residues. That’s why these methods give the results consistent among themselves and with characteristics of evaluation the physical properties of gluten.

SDS 1 is the most informative method for determining physical properties of gluten (see Table. 3, 4). Indicators of sedimentation in acetic acid (Pumpyansky-Sozinov) were correlated with grain-unit (see Table. 3). In 2008,  a small but significant correlation between grain size and the level of destruction by chinch was observed, as well as the correlation between IDC and the content of wet and dry gluten (see Table. 3, 4).

To estimate the relative content of cysteine residues (C) involved in polypeptides aggregation using sedimentation rates obtained by SDS 1 and SDS 2 methods, the difference between these indices (SDS 1 -   SDS 2) can be divided by the content of dry gluten or protein expressed as a percentage.

The analysis has revealed a significant negative correlation between C and IDC, the amount of dry and wet gluten (see Table. 3), but no correlation with the level of destruction by grain chinch, grain weight and grain-unit. The samples of varieties Selyanka odesskaya and Uni 1 showed the highest C rates (6,52 and 6,11). Selyanka odessdaya, according to investigators from Odessa, refers to superpower wheat (14). The varieties of this type are known, and the reasons for their high quality are described. For example, the superpower wheat variety Red River has a tandem duplication in the allele Glu-B1al which controls the synthesis of high-molecular glutelins which participate in aggregation of protein molecules through disulfide bonds (10, 11). The superpower variety Bànkúti 1201 possesses the allele Glu-1Ah2 * b (15) with substituted basis in the position 1181, which leads to the replacement of serine by cysteine in gluteline molecule. The additional cysteine residue leads to a raise of aggregation, which improves physical properties of gluten and bread baking qualities of flour.

At the same time, applying electrophoresis can’t help to distinguish the mutant protein controlled by the allele Glu-1Ah2*b  from the protein of wild type (the allele Glu-1Ah2*)  by its molecular weight and charge.

The sample Uni 1 has a specific feature - the absence in its endosperm of ω-gliadins controlled by chromosome 1D (12). These proteins contain no cysteine, which leads to the increase in proportion of other polypeptides in weevil endosperm and the improved flour quality compared with those in initial forms. The absence of ω-gliadins can be easily tested by electrophoresis.

The lowest C rate (Table 5) was detected in the variety Synthetic carrying 1B.1R translocation. The rye chromosome 1R controls the synthesis of sekalins – endosperm storage proteins. Would rye proteins be synthesized in wheat, they adversely affect baking properties of wheat flour (13), because wheat proteins and rye sekalins are unable to form the highly-polymeric insoluble protein complexes providing gluten with high quality physical characteristics (16). Thus, the presence of rye proteins in wheat endosperm reduces the number of intermolecular disulfide bonds. The variety Bogdanka carries rye translocation as well (in the 1A chromosome (1AL.1RS). Bogdanka demonstrates a lower C rate (0,94) (see Table. 5), though, this value is higher than that of the sample Synthetic. Consequently, the degree of worsening the wheat flour quality may depend on which of the homologous chromosomes (1A, 1B or 1D) carries the translocated arm 1RS.

The gene for soft grain (Soft) compared with the gene for durum grain (hard) leads to the significant decrease in flour swelling owing to starch complex rather than gluten protein (17). Indeed, the winter soft variety Volga 100 corresponding to soft grain type differed from other varieties (see Table. 5): in year 2008, Volga 100 demonstrated the values of SDS 1 and of Pumpyansky-Sozinov amounted to 51.0 and 23 5 ml resp. (averages for the experiment - 68.2 and 29.8 ml, resp). Therefore, a soft grain property reduced these rates to 22-25%, and C rate in Volga 100 (2,78) seem to be the correct characteristic for the number of intermolecular disulfide bonds in endosperm storage proteins.

Since measuring C requires a time consuming procedure for determining the amount of protein or dry gluten content in grain, authors performed a correlation analysis between the value of C and the difference (Δ) between SDS 1 and SDS 2 in 68 samples to obtain r = 0,9847 ± 0,0214 (t = 45,96). The determination coefficient between these indices amounted to 96.97%. Consequently, for the mass evaluation of quality, it is sufficient to analyze the samples’ sedimentation rates in the variants SDS 1 and SDS 2.  In the varieties possessing rye translocation 1BL.1RS and 1AL.1RS, Δ was  -2,7 and +8.3 ml, resp., in superpower samples - 54,8 - 63,5 ml, resp.  The sample 252/08 obtained by crossing the varieties Belgorodskaya 12 and Selyanka odesskaya showed C equal to 7,06, which was  appropriate to the value of the parent form Selyanka odesskaya (see Table. 5). According to the data of IDC-test (2007-2008), this sample demonstrated the gluten of I quality group, which confirms its high rates.

Thus, considering various reasons leading to the increase in number of intermolecular disulfide bonds, as well as difficulties in electrophoretic identification of  certain polypeptides, it becomes necessary to develop the integrated approaches to quantitative assessment of cereal proteins. Authors suppose that the proposed modifications of SDS-method providing relative estimation of the number of intermolecular disulfide bonds (C) allows to obtain the resulting characteristic of gluten quality connected with presence in the endosperm of cysteine-keeping polypeptides. This approach can be used for identification of forms with increased number of intermolecular disulfide bonds among the collection material and hybrid offspring.

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