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PubMed2005

Towards the rational design of cereal starches.

A major challenge in cereal biotechnology is to achieve the rational design of renewable polymers to meet specific requirements for improving human health, nutrition, and food quality, to increase the energy supply, and to provide safer and more profitable industrial inputs. The field of starch synthesis research has advanced at a rapid pace over the past decade, and many core observations about the pathway are well established over a range of species. Owing to the complexity of the starch-synthesis process, in which suites of enzymes act at the interface between soluble and insoluble phases, the rational design of starch granules with specific functionality is still in its infancy. Our fundamental biochemical knowledge of starch biosynthesis has recently advanced, and this new information could be exploited to create novel variability in carbohydrate polymers in cereal grains. We propose two strategies for moving more rapidly towards truly rational design of starch. First, the focusing of fundamental research on processes that are involved in the regulation of starch synthesis and granule assembly. Second, the development of iterative strategies, exploiting new molecular genetics tools, to screen for desired properties in high-throughput systems.

Biotechnology↗
PubMed2004

Influence of agricultural practices on fusarium infection of cereals and subsequent contamination of grain by trichothecene mycotoxins.

Fusarium head blight (FHB) of small grain cereals and ear rot in maize are significant diseases across the world. Infection can not only result in reduced yield as a result of shrunken grains but also result in reduced milling and malting quality and the contamination of grains with mycotoxins. Mycotoxins are hazardous to animal and human health. Therefore, guidelines and legislation are in place, or under consideration, in most countries to protect consumers and animal welfare. As fusarium mycotoxins are produced within the growing crop, it is important to understand how agricultural practices affect mycotoxin contamination of grain. Such information could then be used to determine guidelines on "Good Agricultural Practice" (GAP) to minimise the mycotoxin contamination of cereal products. Evidence is provided to show the importance of choice of cultivar, crop rotation, soil cultivation, fertiliser and the chemical and biological control of insects, weeds and fungi.

Agriculture↗
PubMed2000

Simultaneous determination of trichothecene mycotoxins and zearalenone in cereals by gas chromatography-mass spectrometry.

Trichothecene mycotoxins are commonly distributed in crop fields in the world, and zearalenone has been found in cereals. Rapid and accurate methods for the determination of these mycotoxins is required to prevent the intoxication of humans, and to contribute to the supply of safe foods and feeds for human and livestock. Gas chromatography-mass spectrometry (GC-MS) is a useful method for the determination of these toxins. We describe here our current GC-MS analysis for the simultaneous determination of trichothecenes and zearalenone contaminants in cereals.

Edible Grain↗
PubMed1983

Separation of cereal proteins by reversed-phase high-performance liquid chromatography.

Cereal proteins have been extremely difficult to purify and characterize owing to their heterogeneity, poor solubility and tendency to polymerize. High-performance liquid chromatography (HPLC) on a 300 A reversed-phase (RP)(C18) support (Syn Chropak RP-P), using acetonitrile as organic modifier in the presence of trifluoroacetic acid, has been found to be capable of high-resolution separations of these proteins; the resolution is often better than that obtained by any other chromatographic or electrophoretic method. Examples are presented showing separations of low-molecular-weight gliadins, omega-gliadins and ethanol-soluble reduced glutenin subunits from wheat and of zein from corn. In addition, proteins may be directly extracted from ground single kernels and subsequently analyzed by RP-HPLC; applications in genetic studies, in breeding programs and in varietal identification are proposed. In addition to its high resolution, RP-HPLC is superior to most other methods in speed, sensitivity, reproducibility and suitability for quantitation. Polypeptide chains of molecular weight up to 133,000 are recovered in high yields and the column capacity is high, demonstrating that RP-HPLC is suitable for both preparative and analytical separations of proteins. RP-HPLC resolves proteins primarily on the basis of differences in surface hydrophobicity, so it therefore complements, rather than duplicates, other techniques that separate proteins on the basis of size or charge. RP-HPLC promises to become an invaluable technique for the fractionation and characterization of proteins from cereals and other sources.

Chromatography, High Pressure Liquid↗
PubMed2003

Determination of T-2 toxin in cereal grains by liquid chromatography with fluorescence detection after immunoaffinity column clean-up and derivatization with 1-anthroylnitrile.

1-Anthroylnitrile (1-AN) has been shown to be an efficient labelling reagent for the determination of T-2 toxin (T-2) by high-performance liquid chromatography (HPLC)-fluorescence detection. This reaction has been used to develop a sensitive, reproducible and accurate method for the determination of T-2 in wheat, corn, barley, oats, rice and sorghum. The method uses immunoaffinity columns containing antibodies specific for T-2 for extract clean-up, pre-column derivatization with 1-AN and HPLC with fluorescence detection for toxin determination. Ground cereal samples were extracted with methanol-water (80:20, v/v), the extracts were purified by immunoaffinity columns and the toxin was quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 381 nm, emission wavelength 470 nm) after derivatization with 1-AN. Recoveries from the different cereals spiked with T-2 at levels ranging from 0.05 to 1.5 microg/g were from 80 to 99%, with relative standard deviations of less than 6%. The limit of detection was 0.005 microg/g, based on a signal-to-noise ratio of 3:1.

Anthracenes↗
PubMed1996

Determination of cereal herbicide residues in environmental samples by gas chromatography.

Gas chromatographic analysis of cereal herbicide residues in water, soil, plant and air is reviewed. Herbicides widely used in spring and winter cereals, i.e., phenoxyacids, benzonitriles, ureas, triazines, dinitroanilines, chloroacetamides and thiocarbamates, are considered. The main procedures used in the residue analysis, extraction, clean-up, derivatization and gas chromatographic determination are summarized and discussed.

Chromatography, Gas↗
PubMed1998

Liquid chromatographic method for determining added folic acid in fortified cereal products.

Reversed-phase ion-pair high-performance liquid chromatography (HPLC) was coupled with detection by UV absorption (280 nm) for separation and quantitation of added folic acid (FA) in fortified cereal based foods. A simple and rapid liquid-solid extraction method, combined with enzymatic digestion, to recover FA from the sample matrices is also presented. The quantitation of added FA was achieved in products including corn (maize), wheat-, rice- and oat-based cereal breakfast foods fortified at 25% and 100% of the reference daily intake (RDI). The retention time for FA was ca. 15 min, and the detection limit was 2 ng/20 microliters injection for standard FA. When FA was added to unfortified samples of wheat flour at concentrations of 3.08 or 20.0 micrograms/g, recoveries were 93% and 96%, respectively. Comparison of HPLC results with those of a standard microbiological assay has shown quite good agreement (r = 0.998). A solid-phase extraction clean-up procedure has also been developed for use with samples fortified with low levels of FA, where interferences may otherwise hinder quantitation.

Avena↗
PubMed2003

Saponins in cereals.

Saponins are a diverse family of secondary metabolites that are produced by many plant species, particularly dicots. These molecules commonly have potent antifungal activity and their natural role in plants is likely to be in protection against attack by pathogenic microbes. They also have a variety of commercial applications including use as drugs and medicines. The enzymes, genes and biochemical pathways involved in the synthesis of these complex molecules are largely uncharacterized for any plant species. Cereals and grasses appear to be generally deficient in saponins with the exception of oats, which produce both steroidal and triterpenoid saponins. The isolation of genes for saponin biosynthesis from oats is now providing tools for the analysis of the evolution and regulation of saponin biosynthesis in monocots. These genes may also have potential for the development of improved disease resistance in cultivated cereals.

Biological Evolution↗
PubMed1993

Fusarium poae and Fusarium sporotrichioides in cereal grains of the Slovak Republic.

The occurrence of Fusarium poae and F. sporotrichioides was examined in 37,172 cereal grains of 229 samples from the Slovak Republic. In an area where strains of Fusarium parasitized. 40% of grains on average, owing to unfavourable weather, 5 strains of F. poae were isolated (0.5% out of 1,015 strains of Fusarium). In an area where the situation was less unfavourable, Fusarium spp. contaminated on average 4.1% of grains, F. poae were present in 6.1% out of 82 strains of Fusarium. When cereal ripened and harvested under climatically favourable conditions. Fusarium spp. were isolated, on average, out of 3.0% of grains, but the strains of F. poae made an essential part of the mycoflora (25.5% out of 985 isolated Fusarium strains). In three samples there were ascertained 5%, 15% and 19.5% of grains contaminated by strains of F. poae, F. sporotrichioides was isolated only 5 times.

Climate↗
PubMed1998

Group specific PCR-detection of potential trichothecene-producing Fusarium-species in pure cultures and cereal samples.

A PCR based assay (Tox5 PCR) which analyses Fusarium species potentially producing trichothecenes was developed using a pair of primers derived from the DNA-sequence of the trichodiene synthase gene (tri5). The primer pair was tested using DNA isolated from a variety of strains representing 64 species and varieties of Fusarium as well as from other fungi, bacteria and cereals. A 658 bp PCR fragment was specifically amplified with DNA isolated from strains of species belonging to the Fusarium sections Discolor, Sporotrichiella, Arthrosporiella, Gibbosum, and "Dlaminia". PCR products obtained were sequenced. Alignment to tri5 sequences given in the literature revealed a high degree of homology. Results of the PCR developed correlated well with literature data on the trichothecene producing capabilities of the respective species. Potential trichothecene producing fusaria were detected in contaminated cereals and malts using the Tox5 PCR assay. Intensity of the signals produced were well correlated with the concentration of deoxynivalenol (DON) in samples of wheat.

Base Sequence↗
PubMed1996

Cereal genome analysis using rice as a model.

Researchers are eagerly waiting for the physical map of rice to become completed and available for use as a model for all cereals. The most significant advances of the past year have been the progress toward positional cloning of genes and the identification of quantitative trait loci (QTL) from detailed restriction fragment length polymorphism maps. Future focus will be: first, the enhanced dissemination and integration of the available data in World Wide Web accessible databases for easy comparison of genetic and physical mapping data across various species; second, the expanded distribution of a wide variety of DNA materials (cDNA clones, yeast artificial chromosomes, bacterial artificial chromosomes and other probes) for use in other cereals on the basis of the rice model map; and third, the applied breeding by locating and isolating sequences corresponding to important agronomic traits, often correlating with QTL.

Chromosome Mapping↗
PubMed1998

Advances in cereal gene transfer.

Over the past five years, transgenic strains of various major cereals have been produced, with transformation of rice and maize being most common. A majority of the cereal transformants obtained to date has been generated by the particle bombardment technique, but Agrobacterium-mediated transformation is rapidly becoming the method of choice. Rice, the plant in which transformation-related technology is most advanced, appears to be the model monocotyledon for basic and applied studies.

Edible Grain↗
PubMed2003

Analysis of rDNA ITS sequences to determine genetic relationships among, and provide a basis for simplified diagnosis of, Fusarium species causing crown rot and head blight of cereals.

Genetic relationships of the complex of Fusarium species associated with crown rot and head blight in cereals and some species associated with plant diseases in general were examined by distance and maximum parsimony algorithms of their internal transcribed spacer sequences. The analysis clustered the complex of Fusarium species that causes root and crown rot and head blight of cereals and three other clusters of F. sambucinum, F. venenatum and F. poae into one clade. This group of Fustarium species was also found in this study to correspond to the group defined by the presence of the tri5 gene. The tri5 gene was recently reported to co-segregate with the locus governing the type of trichothecene produced, and probably maps in the trichothecene gene cluster. The other clusters of F. avenaceum, F. tricinctum, F. torulosum, F. oxysporum, F. verticillioides and F. solani did not have the tri5 gene. Although, F. pseudograminearum was phylogenetically close with the cluster of F. graminearum, F. culmorum and F. cerealis, it could be distinctly separated from them. The distinct genetic status of F. pseudograminearum from F. graminearum corroborated with other published molecular data and isozyme findings. The molecular analysis provided a simple diagnostic tool to differentiate fungi causing crown rot from those involved in head blight.

Cluster Analysis↗
PubMed2000

Detection of T-2 toxin in different cereals by flow-through enzyme immunoassay with a simultaneous internal reference.

In most previously described membrane-based immunoassays a separate negative control assay is always carried out to evaluate the performance of the assay. To overcome this problem, a membrane-based flow-through enzyme immunoassay with an internal control has been developed for the detection of T-2 toxin in cereals (patent pending). An Immunodyne ABC membrane was coated with 2 microL of goat anti-horseradish peroxidase (HRP) (internal control spot) (1:1000) and 2 microL of rabbit anti-mouse (test spot) (undiluted) immunoglobulins, and the free binding sites were blocked. In addition to the antibody-coated Immunodyne ABC membrane, the assay also comprises a plastic snap-fit device, absorbent cotton wool, mouse anti-T-2 monoclonal antibodies (Mab), and T-2-HRP conjugate. The color intensity (Delta) of the internal control and that of the negative sample showed no difference (P > 0.05), whereas there was a significant difference between the internal control and positive samples (P < 0.05). The minimum detectable limit that could be visually detected with confidence was 50 ng of T-2 per gram of cereal sample.

Edible Grain↗
PubMed2002

Application of manual and automated systems for purification of ochratoxin a and zearalenone in cereals with immunoaffinity columns.

A manual vacuum manifold and an automated solid phase extraction (ASPEC) system were applied for purification of ochratoxin A and zearalenone in wheat, rye, barley, and oat samples with immunoaffinity columns followed by separation with a high-performance liquid chromatograph and fluorescence detection. The immunoaffinity columns for manual sample purification were purchased from a different manufacturer than were those for the automated system. The limit of detection (LOD) for the method for ochratoxin A with a vacuum manifold and ASPEC was 0.1 microg/kg. For the method for zearalenone, the LODs were 1.5 microg/kg with a vacuum manifold and 3 microg/kg with ASPEC. For the methods for ochratoxin A at spiking levels of 0.6 and 2.5 microg/kg, mean recoveries for different cereals varied from 68 to 106%. For the methods for zearalenone, mean recoveries varied from 78 to 117% at spiking levels of 9 and 25 microg/kg. The relative standard deviations of repeatability with various cereals employing both methods were 2-15 and 2-19% for ochratoxin A and zearalenone, respectively.

Carcinogens↗
PubMed2002

Near-infrared analysis of soluble and insoluble dietary fiber fractions of cereal food products.

The use of near-infrared (NIR) reflectance spectroscopy for the rapid and accurate measurement of soluble and insoluble dietary fiber was explored in a diverse group of cereal products. Ground samples were analyzed for soluble and insoluble dietary fiber (AOAC Method 991.43) and scanned (NIRSystems 6500 monochromator) to obtain NIR spectra. Modified PLS models were developed to predict insoluble and soluble dietary fiber using data sets expanded to include products with high fat and high sugar contents. The models predicted insoluble dietary fiber accurately with an SECV of 1.54% and an R(2) of 0.98 (AOAC determined range of 0-48.77%) and soluble dietary fiber less accurately with an SECV of 1.15% and an R(2) of 0.82 (AOAC determined range of 0-13.84%). Prediction of independent validation samples by the soluble fiber model resulted in a bias that may be related to the way the reference method treats samples with different soluble fiber constituents. The insoluble fiber model can be used to rapidly monitor insoluble dietary fiber in cereal products for nutrition labeling.

Calibration↗
PubMed2003

Determination of the activity of acidic phytate-degrading enzymes in cereal seeds.

Five different methods were compared to elucidate the total activity of the acidic phytate-degrading enzymes present in the seeds of rye, wheat, and barley. Phytate-degrading activity was studied at pH 5.0 by quantifying the liberated phosphate. Rye showed the highest acid phytate-degrading activity among the cereals studied. Using an aqueous extract, only 30-50% of the activity was found (rye, 3443 mU g(-1) of grain; wheat, 1026 mU g(-1) of grain; barley, 1032 mU g(-1) of grain) compared to that found by the direct incubation of the dry-milled cereal grains in a buffered phytate-containing solution (rye, 6752 mU g(-1) of grain; wheat, 2931 mU g(-1) of grain; barley, 2093 mU g(-1) of grain). Extending the extraction time resulted in an increase in extractable phytate-degrading activity by, at maximum, 10-15%. Extraction of phytate-degrading activity is strongly enhanced in the presence of Triton X-100 and the protease inhibitor phenylmethylsulfonyl fluoride (rye, 6536 mU g(-1) of grain; wheat, 2873 mU g(-1) of grain; barley, 2023 mU g(-1) of grain), suggesting at least a partial association with membrane structures and a degradation by proteolytic activity during extraction. In addition, it was shown that determining phytate-degrading activity by quantification of the liberated inorganic phosphate is more robust and precise than determining phytate-degrading activity by quantification of the residual phytate.

6-Phytase↗
PubMed2003

Sinapate dehydrodimers and sinapate-ferulate heterodimers in cereal dietary fiber.

Two 8-8-coupled sinapic acid dehydrodimers and at least three sinapate-ferulate heterodimers have been identified as saponification products from different insoluble and soluble cereal grain dietary fibers. The two 8-8-disinapates were authenticated by comparison of their GC retention times and mass spectra with authentic dehydrodimers synthesized from methyl or ethyl sinapate using two different single-electron metal oxidant systems. The highest amounts (481 microg/g) were found in wild rice insoluble dietary fiber. Model reactions showed that it is unlikely that the dehydrodisinapates detected are artifacts formed from free sinapic acid during the saponification procedure. The dehydrodisinapates presumably derive from radical coupling of sinapate-polymer esters in the cell wall; the radical coupling origin is further confirmed by finding 8-8 and 8-5 (and possibly 8-O-4) sinapate-ferulate cross-products. Sinapates therefore appear to have an analogous role to ferulates in cross-linking polysaccharides in cereal grains and presumably grass cell walls in general.

Coumaric Acids↗