CONTENTS Chapter 1: Cereal Foods in Diet and Health.........................................................1 Kaisa Poutanen Chapter 2: Sterols ...................................................................................................7 Laura Nyström, Tanja Nurmi, Anna-Maija Lampi, and Vieno Piironen Chapter 3: Tocopherols and Tocotrienols...........................................................15 Anna-Maija Lampi and Vieno Piironen Chapter 4: Analytical Procedures for Determination of Alk(en)ylresorcinols in Cereals and Cereal Products....................................25 Rikard Landberg, Afaf Kamal-Eldin, Annica A. M. Andersson, and Per Åman Chapter 5: Phenolic Acids ....................................................................................41 Li Li, Claudia Harflett, Michael H. Beale, and Jane L. Ward Chapter 6: Lignan Analysis of Cereal Samples by GC/MS...............................53 Klara Ercsey, Eva Nagy-Scholz, Szilveszter Gergely, and András Salgó Chapter 7: Total Folate ........................................................................................59 Susanna Kariluoto and Vieno Piironen Chapter 8: Carotenoids ........................................................................................69 El-Sayed M. Abdel-Aal and J. Christopher Young Chapter 9: Methods for the Analysis of Selenium and Other Minerals ...........95 Jacqueline L. Stroud, Fang-Jie Zhao, Steve P. McGrath, and Dave Hart Chapter 10: Quantitative Analysis of Oat Avenanthramides ......................... 113 Lena H. Dimberg and Jelena Jastrebova Chapter 11: Phytate ............................................................................................ 129 Erika Skoglund, Nils-Gunnar Carlsson, and Ann-Sofie Sandberg Chapter 12: Anthocyanidins .............................................................................. 141 J. Christopher Young and El-Sayed M. Abdel-Aal Chapter 13: Total Dietary Fiber ........................................................................ 167 Danuta Boros and Per Åman v
vi HEALTHGRAIN Methods: Analysis of Bioactive Components in Small Grain Cereals
Chapter 14: Quantification of Arabinoxylans and Their Degree of Branching Using Gas Chromatography ....................................................... 177 Kurt Gebruers, Christophe M. Courtin, and Jan A. Delcour Chapter 15: Enzymatic Mapping of Arabinoxylan Structure......................... 191 Luc Saulnier and Bernard Quemener Chapter 16: Molecular Weight Distributions of Water-Extractable β-Glucan and Arabinoxylan ............................................................................... 203 Roger Andersson, Annica Andersson, and Per Åman Chapter 17: Spatial Mapping of Cell Wall Components in the Cereal Endosperm Using Spectroscopic, Fluorescent and Immunochemical Methods.......................................................................... 217 Geraldine A. Toole, Nikolaus Wellner, Craig B. Faulds, E. N. Clare Mills, Cecile Barron, Marie Françoise Devaux, and Fabienne Guillon Chapter 18: Screening for Dietary Fiber Constituents in Cereals by Near Infrared Spectroscopy.......................................................................... 247 András Salgó, Szilveszter Gergely, and Kurt Gebruers Chapter 19: Combining Bioactive Components with Conventional Targets in Plant Breeding Programmes............................................................ 263 Zoltan Bedő, Mariann Rakszegi, Laszlo Láng, Kurt Gebruers, Vieno Piironen, Domenico Lafiandra, Jane Ward, Andy Phillips, and Peter R. Shewry Chapter 20: Future Prospects for the Analysis of Bioactive Components in Cereal Grain ............................................................................. 273 Jane L. Ward and Peter R. Shewry Index..................................................................................................................... 281
Chapter 20
FUTURE PROSPECTS FOR THE ANALYSIS OF BIOACTIVE COMPONENTS IN CEREAL GRAIN Jane L. Ward and Peter R. Shewry Centre for Crop Genetic Research, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK INTRODUCTION The HEALTHGRAIN program is one of a number of initiatives to increase the consumption and benefits of wholegrain cereals at the international, national, and local levels. Perhaps the most important of these is the HarvestPlus program of the Consultative Group on International Agricultural Research (CGIAR), which includes increasing iron, zinc, and provitamin A in wheat amongst a range of targets to improve the nutrition of the poor, particularly those in developing countries (http://www.harvestplus.org/) (Ortiz-Monasterio et al., 2007). Similarly, the Australian Food Futures Flagship project of CSIRO (http://www.csiro.au/org/ FoodFuturesFlagship.html) includes grain composition and human health as one of a number of targets in food quality. All of these programs require the analysis of multiple samples in order to identify variation in composition in germplasm, lines from breeding programs, grain fractions, and food products. Although the methods described in this volume are all “tried and tested,” many of them are time-consuming, expensive, and require specialized equipment. Furthermore, separate analyses are required for most groups of compounds, which may each require different skills and equipment. It is clearly necessary to simplify and combine the analyses for bioactive components if they are to become widely used. SIMPLIFICATION OF ANALYSES NIR spectroscopy is widely used in the plant breeding and food industries, providing robust high throughput analyses for grain components such as starch, protein, and water. Chapter 18 in this volume describes how NIR calibrations can be developed for arabinoxylans, which are present at lower levels in the grain. However, these components account for up to 3% of white flour and over 20% of bran and we do not yet know whether robust calibrations can be developed for minor components such as sterols and tocols (less than 100 and 1,000 µg/g wholemeal, respectively) (data from Ward et al., 2008). 273
274 HEALTHGRAIN Methods: Analysis of Bioactive Components in Small Grain Cereals
Other options are to develop specific antibody-based or enzyme-based kits. Considerable success has been achieved in developing and marketing enzyme-based
Fig. 1. Typical 1H NMR spectrum (400 MHz) of wheat flour following extraction by 80:20 D2O:CD3OD. (a) full spectrum; (b) central region of the spectrum, dominated by overlapping carbohydrate signals; (c) aliphatic region of the spectrum dominated by aliphatic amino acids. Reprinted from Baker el al. (2006) with permission.
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kits for major grain components such as β-glucans, total starch, and the proportion of amylose (see, for example, kits marketed by Megazyme, Bray, Ireland), and there is no doubt that this range could be extended if sufficient demand was demonstrated. COMBINING ANALYSES: METABOLOMICS Metabolomics can be defined as a comprehensive analysis where all of the small metabolites present in a tissue are analyzed in an unbiased manner. The ultimate aim is to determine the whole complement of metabolites in a single analysis although this is not currently feasible due to differences in the concentrations and chemical properties of components. In practice two systems are widely used, based on nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) with the sample being introduced directly into the mass spectrometer or after separation by gas or liquid chromatography. These two approaches are to some extent complementary and their principles, advantages, and drawbacks are discussed by Hall (2006) and Ward et al. (2007). Baker et al. (2006) described the application of NMR fingerprinting to determine the substantial equivalence of field-grown, genetically-modified, and parental wheat lines but also used GC-MS for additional quantitative amino acid analyses. This study illustrated the current status in the application of metabolomics technologies to wheat, in that the spectra of fractions extracted with deuterated methanol/ water were dominated by the major soluble components (mainly carbohydrates) with minor peaks corresponding to the most abundant free amino acids (Fig. 1). The only “bioactiveâ€? components which are extracted under these conditions and are present in sufficient amounts to be detected are the methyl donors, choline, and betaine, and NMR therefore provides an excellent system for these to be determined in high throughput analyses (Fig. 2). In our own laboratory, these analyses can be carried out at a rate of six samples/hour using robotics for weighing and extraction and automated injection into a 600 MHz NMR instrument (Fig. 3). The data files from the analysis can also be used for multivariate analysis to compare the fingerprints of different lines, treatments, or fractions (Fig. 4). However, the real challenge for the application of metabolomics technology, whether MS-based or NMR-based, to biologically active components in grain is to develop systems for the simultaneous extraction of multiple groups of components and to remove (either chemically or by data processing) the signals originating from the major components to allow trace amounts of bioactive components to be identified and quantified. Although a full metabolomic analysis of bioactive components may ultimately not be achievable, we are confident this approach will at least be applicable to groups of components with similar chemical properties, for example, terpenoids or phenolic components. EXTRACTION SOLVENTS FOR METABOLOMICS The majority of techniques utilized in this handbook still rely on an initial solvent extraction being made to release the metabolites from the biological tissue and the careful selection of this initial extraction solvent is paramount in dictating the final complement of metabolites present in the final sample. Polar metabolites can be released using isopropanol, ethanol, methanol, acidic methanol, acetonitrile
276 HEALTHGRAIN Methods: Analysis of Bioactive Components in Small Grain Cereals
Fig. 2. Analysis of triplicate samples of 31 cereal bran lines by 1H NMR, demonstrating ability to analyze methyl donors, such as betaine and choline, within a typical NMR fingerprinting screen.
(Aharoni et al., 2002), water, methanol/water (Roessner et al., 2000), or other mixtures of these solvents, while more lipophilic metabolites can be extracted by chloroform or ethyl acetate. The advantage of extracting samples with mixtures of water/methanol/chloroform or ethyl acetate is the generation of a biphasic sample and the fractionation of the metabolites into polar aqueous and lipophilic organic fractions, which can then be analyzed separately (Fiehn et al., 2000). While a large number of studies employ extraction with a single solvent, as outlined above, the key to developing a method for the simultaneous detection of a range of phytochemicals may be to employ a tiered or multi-level extraction strategy and to “stitch” the data together to form a “virtual metabolomic signature.” Although this approach is more complex by design and yields multiple analytical samples per biological sample, it may be the only way to obtain data on a range of diverse metabolites which previously have required either non-polar extractions, polar extractions, or saponification methodologies to release them from the raw material. This approach has recently been applied to the analysis of rice grain with the analysis of four different solvent fractions providing information on a range of metabolites from non-polar lipids to common carbohydrates, amino acids and organic acids through to tocols, phenolics, and sterols. (Shu et al., 2008)
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Fig. 3. NMR laboratory at The National Centre for Plant and Microbial Metabolomics at Rothamsted Research (http://www.metabolomics.bbsrc.ac.uk).
Fig. 4. Principal component analysis (PCA) of 1H NMR data from field grown wheat flour demonstrating ability of the technique to cluster samples from different environments and harvest times. Reprinted from Baker el al. (2006) with permission.
278 HEALTHGRAIN Methods: Analysis of Bioactive Components in Small Grain Cereals
INSTRUMENTATION The most successful metabolomics studies employ a broad range of analytical techniques and integrate the data to give the broadest coverage of the metabolome. For quantitation of metabolites with a wide range of polarities, LC-MS may offer the best way forward. The use of modern multi-sector instruments, set up to monitor multiple target compounds by using the technique of multiple reaction monitoring (MRM), provides significantly increased sensitivity and selectivity by effectively screening out all of the “unwanted” ions and concentrating on only the metabolite signals of interest (Kitteringham et al., 2008). Coupling this system with ultra performance liquid chromatography (UPLC) technologies would speed up sample throughput by shortening chromatography run times and thus make the technology amenable to high throughput screens of large sample numbers such as quality trait analysis or complex genotype × environment (G × E) experiments. While many studies use combinations of GC-MS or LC-MS, the use of mass spectrometry without prior chromatography is becoming increasingly popular and provides a fast screening method for all of the ionizable metabolites present in the sample. This technique has been successfully used to profile a number of different biological systems including, more recently, polyphenolics in fruit (Stewart et al., 2007). The use of an initial polar solvent extraction makes it possible not only to capture information on primary metabolites such as carbohydrates and amino acids but also to determine changes in secondary metabolites such as phenylpropanoids, anthocyanins, and flavonoids, which are antioxidant molecules present in plant tissues including cereals. Fourier transformation ion cyclotron resonance mass spectrometry (FT-ICR-MS) provides an extension to the “nominal mass” direct infusion technique and is capable of producing a fingerprint of unparalleled resolution and accurate mass data which is sufficient to confidently suggest chemical formulae and thus help to identify the large number of “unknowns” present in any typical metabolomics sample (Brown et al., 2005). The restriction, however, is that unlike other techniques, FT-MS cannot distinguish between isomeric compounds and may need to be coupled to liquid chromatography to achieve this. Laser desorption/ionization (LDI) MS and matrix assisted laser ionization/desorption (MALDI) MS have not been widely used in plant metabolomics to date generally because of difficulties in generating ions from the relatively hydrophobic plant tissue and the problems of utilizing a chemical matrix which itself gives rise to many low molecular weight ions. That said, a number of successful studies using these techniques have been reported. In cereals, MALDI-MS imaging has recently been used to profile a variety of metabolites in wheat seeds using a conventional matrix (Burrell et al., 2007) whilst LDI MS imaging approaches have been used for the direct profiling of plant carotenoids (Fraser et al., 2007), bark tannins (Ishida et al., 2005), phosphatidylcholine (Zabrouskov et al., 2001) and, via the use of graphite as an assisting material (GALDI MS), lipids, organic acids, and flavonoids from plant tissues (Zhang et al., 2007; Cha et al., 2008). The obvious advantage of these techniques is their ability to spatially profile metabolites in intact tissues without the need for an extraction protocol which will inevitably discriminate against certain classes of chemical compound. An extension of the philosophy of analysis without extraction is further demon1 strated by the utilization of H NMR analysis of the intact cell wall, via solubilization, to yield important information on the difficult to analyze cell wall bound phe-
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nolics, lignin, and fiber components (Yelle et al., 2008). Although in its infancy, this technique obviously offers the potential to analyze a large number of biologically important metabolites, using one technique, which have previously relied upon a variety of methods involving, in some cases, a lengthy and complex sample preparation protocol. CONCLUSIONS In recent years there has been much development of analytical instrumentation with new equipment offering increased sensitivity, dynamic range, and detection limits. While this no doubt will assist in the analysis of complex mixtures of bioactive compounds, there remains the problem of trying to attain the holy grail of encapsulating all of the desired information on a range of complex phytochemicals in a single analytical run. It is not anticipated that this goal will be reached in the very near future, but developments in sample preparation strategies, the use of multiple extraction procedures, or the utilization of techniques which bypass solvent extraction completely, may increase number and diversity of the metabolites under study in any given sample and this, coupled with the ever evolving and advancing technology, whether mass spectrometry or NMR, may allow researchers, particularly those involved in the analysis of bioactive components in cereals, to combine and simplify their methods, whilst increasing the range of components analyzed. ACKNOWLEDGEMENTS This publication is financially supported by the European Commission in the Communities 6th Framework Programme, Project HEALTHGRAIN (FOOD-CT-2005-514008). It reflects the authors’ views and the Community is not liable for any use that may be made of the information contained in this publication. Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the UK.
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