Value Added Wheat CRC and Food Science Australia, North Ryde, NSW 1670, Australia
Decades ago, wheat was considered a bulk commodity with minor regard neither for differences in quality type nor for market requirements. However, these attitudes have changed dramatically, and now specific quality grades are segregated at harvest. Each grade is designed to meet the quality specifications of specific niche markets, such as Udon noodle manufacture in Japan, flat-bread production in Middle Eastern countries and yellow alkaline noodles in Korea. However, a major limitation to these attempts to add value to the wheat harvest has been the lack of suitable methods to test grain for relevant aspects of quality under the rushed conditions of grain receival at harvest.
Several recent developments in the Australian wheat industry have increased the significance of genotypic characteristics and the need for adequate methods of determining varietal identity. The implementation of Plant Breeder's Rights requires that varietal identity must be verified for grain deliveries, in relation to the payment of End-Point Royalties. In addition to the long-standing system of requiring that only specific varieties are permitted for delivery into most grades, AWB Ltd has recently introduced the additional system, known as “Golden Rewards Premium Choice”, whereby selected varieties receive premium payments in allocated classification regions under the AWB “National Pool Golden Rewards” program.
The preservation of identity of grain has the potential to increase the value of the wheat crop, provided that grain of similar quality can be identified and segregated at the receival stage. The first step in this process is to effectively identify variety and/or quality type ‘on-the-spot’ under the rushed conditions of the country elevator and regional laboratory at harvest time. Visual identification has been found to have limited application, being subjective and poor in its ability to discriminate. On the other hand, analysis of grain-protein composition has proved to be effective, though slow. For some decades, gel electrophoresis has been the traditional method of protein analysis. As an alternative, reversed-phase high-performance liquid chromatography has also been used. Both these methods are slow and confined to the laboratory, because of the complexity of their use, and of need for back-up resources and trained operators. The Lab-on-a-chip version of capillary electrophoresis promises to overcome these difficulties, thereby offering a platform for quickly identifying grain varieties beyond the confines of the traditional laboratory.
Materials and methods
Authentic samples of wheat varieties were used as a basis for evaluating the capabilities of the Agilent Lab-on-a-chip equipment for distinguishing between varieties of wheat. In addition, multi-null lines lacking specific high-molecular-weight (HMW) glutenin subunits (Lawrence et al 1987) were used to identify peaks in the glutenin-subunit profiles. Breeders’ lines were also used to evaluate the system. Milled wholegrain or flour samples (10 mg) were extracted with 0.5 mL 1% SDS solution containing 1% dithiothreitol (DTT). Ten clarified extracts (4 μL each) were applied with Agilent sample buffer to each of the ten sample wells of a Protein
Figure 1. The Agilent 2100 Bioanalyzer, on the right of the laptop computer. The analysis module is open, showing the position for placing the loaded chip.
200+ LabChip for analysis in the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) following the manufacturer’s instructions (Figure 1).
Distinction between wheat varieties was readily provided by the extraction of all polypeptides, followed by a one-minute run on the 2100 Bioanalyzer, using the Protein 200+ chip (Figure 2). The analysis of the full set of ten samples on a chip takes 30 minutes. The minimum analysis time is about 15 minutes if only one sample is identified, including extraction, centrifugation and analysis. Routinely, however, a set of ten samples would be extracted together and analysed sequentially on the chip, taking about 40 minutes for the full set. These times are considered close to the requirements of grain-receival conditions, when verification of variety may be needed prior to assignment of a load to a specific storage cell. The Lab-on-a-chip fractionates on the size-based system of capillary electrophoresis. Distinction between wheat varieties was effective for all of a set of fifty Australian wheats, as illustrated in Figure 2 for six varieties.
Figure 2. Lab-on-a-chip capillary electrophoresis of wheat-flour proteins extracted under reducing conditions (1% SDS + 1% DTT), and exhibited as elution profiles.
Figure 3. Lab-on-a-chip capillary electrophoresis of glutenin subunits.
Figure 4. PatMatch screen, showing the homology of the test sample’s profile to those of many other varieties. The high similarity of Dollarbird to the test sample indicates its identity.
Due to the relatively large size of the HMW subunits of glutenin, they are well separated from all other polypeptides, appearing last in the profiles (Figure 2) and at the top of the simulated gel patterns (Figure 3). The mobilities of the common HMW subunits have been characterised (numbered in Figure 3), permitting them to be automatically identified by appropriate software. This information offers the opportunity for going beyond the identification of variety to predict dough properties, based on relationships such as the Glu-1 score (Payne, 1987). Alternatively, both high- and low-molecular-weight subunits of glutenin could be analysed by a more complex extraction procedure, involving preliminary extraction to remove non-glutenin proteins (Uthayakumaran et al 2004). The Protein 200 Plus LabChip is best suited to the separation of the HMW subunits together with the LMW subunits if the full range of glutenin subunits is applied.
An advantage of capillary electrophoresis, compared to conventional gel electrophoresis, is that the results are immediately available in digital form, without the extra step of scanning a gel. To take advantage of this, the program PatMatch (Bekes et al 1991) has been adapted to the Agilent system, thereby permitting the output for a test sample to be matched against a library of profiles for authentic samples. The resulting screen (Figure 4) provides a list of the varieties with greatest similarity to the test sample, including the % similarity of the test sample to each variety. In the case shown in Figure 4, the test had 95.7% to Dollarbird. As the variety with the next level of similarity (Janz, 53% similar) is well distant, the identification of the test sample can be confidently given as Dollarbird.
The acidic polyacrylamide gel electrophoresis, high performance liquid chromatography and the capillary electrophoresis has been used for several years to analyse gliadin proteins and the HMW subunits of glutenin. These applications have involved the use of conventional equipment that requires time or a reasonable degree of operator experience and/ or involves large equipment and considerable capital expense. The Lab-on-a-chip overcomes these hurdles, being offered at more modest capital cost, being much smaller, and having automatic loading. The Agilent system is thus a distinct improvement on all other methods for the fractionation of glutenin subunits, providing rapid analysis with immediate interpretation of results using equipment that is small and simple to use. Its portability offers the possibility of using the equipment outside of the conventional laboratory. In the coming wheat harvest, it will be deployed at country silos as a demonstration to growers that variety identification can be and is being performed on deliveries.
Lab-on-a-chip capillary electrophoresis offers a rapid means of testing grain for variety, and also quality type. Its small size enables the transfer of method out of the conventional laboratory.
Bekes, F., Batey, I.L., Wrigley, C.W., and Gore, P.J. (1991). Gluten Proteins 1990, Proc. 4th International Workshop on Gluten Proteins., 467-475.
Lawrence, G.J., Moss, H.J., Shepherd, K.W., and Wrigley, C.W. (1987). J. Cereal Sci. 6, 99-101.
Payne, P. I. (1987). Ann. Rev. Plant Physiol. 38, 141-153.
Uthayakumaran, S., Batey, I.L., and Wrigley, C.W. (2004). Cereals 2004. Proc. 54th Aust. Cereal Chem. Conf., 411-414.