Maize Extracts Research Articles

The determination of avidin in genetically modified maize by voltammetric techniques

Quality assurance is a major issue in the food industry. The authenticity of food ingredients and their traceability are required by consumers and authorities. Plant species such as barley (<i>Hordeum vulgare</i>), rice (<i>Oryza sativa</i>), sunflower (<i>Helianthus annus</i>), wheat (<i>Triticum aestivum</i>) and maize (<i>Zea mays</i>) are very common objects of interest of genetic modification (GMO); therefore the development of specific assays for their specific detection and quantification of GMO are needed. Furthermore, the production and trade of genetically modified lines from an increasing number of plant species brings about the need for control within research, environmental risk assessment, labeling-legal, and consumers’ information purposes. Electrochemical sensors and biosensors based on modification of working electrode could be suitable tools for these purposes. Here, we report using of an avidin-modified carbon paste electrode for rapid and sensitive determination of avidin in plant extract solution and in a transgenic maize extract. The process could be used to determine avidin concentrations up to 3pM in solution and 170nM in a maize seed extract. Moreover, we applied the method to analyze different maize flours.

Metabolism of indole-3-acetic acid in rice: Identification and characterization of N-β- d-glucopyranosyl indole-3-acetic acid and its conjugates

A search was made for conjugates of indole-3-acetic acid (IAA) in rice ( Oryza sativa) using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) in order to elucidate unknown metabolic pathways for IAA. N-β- d-Glucopyranosyl indole-3-acetic acid (IAA- N-Glc) was found in an alkaline hydrolysate of rice extract. A quantitative analysis of 3-week-old rice demonstrated that the total amount of IAA- N-Glc was equal to that of IAA. A LC–ESI–MS/MS-based analysis established that the major part of IAA- N-Glc was present as bound forms with aspartate and glutamate. Their levels were in good agreement with the total amount of IAA- N-Glc during the vegetative growth of rice. Further detailed analysis showed that both conjugates highly accumulated in the root. The free form of IAA- N-Glc accounted for 60% of the total in seeds but could not be detected in the vegetative tissue. An incorporation study using deuterium-labeled compounds showed that the amino acid conjugates of IAA- N-Glc were biosynthesized from IAA-amino acids. IAA- N-Glc and/or its conjugates were also found in extracts of Arabidopsis, Lotus japonicus, and maize, suggesting that N-glucosylation of indole can be the common metabolic pathway of IAA in plants.

Chapter 21 - Assay of Editing of Exogenous RNAs in Chloroplast Extracts of Arabidopsis, Maize, Pea, and Tobacco

Nucleotides within transcripts of chloroplasts and mitochondria are modified through C-to-U RNA editing in vascular plants. The specific protein components and enzymatic machinery required for editing have not been defined. A consensus sequence is not present around all editing sites, complicating the discovery of cis-sequence elements critical for editing. Chloroplast extracts capable of carrying out editing in vitro along with precise quantification of editing extent of exogenous transcripts will facilitate identification of both cis and trans factors. We have optimized an in vitro assay originally developed to study editing in tobacco and pea chloroplasts and have expanded the assay to include the study of chloroplast editing in the model species Arabidopsis and the monocot maize. The superior genetic resources in these two species can now be utilized in conjunction with biochemical analysis to dissect the editing apparatus. We have improved the assay conditions for editing in vitro, achieving efficient editing (as much as 92%) with certain RNA substrates. Unlike the initial assay that relied on qualitative analysis, we are able to achieve precise quantification of editing activity within 1% through a simple poisoned primer extension (PPE) assay with radiolabeled oligonucleotides.

International shipping of fumonisins from maize extracts on C18 sorbent

Fumonisins are mycotoxins found in maize. In developing countries, the resources required for analysis are often lacking, and the shipping of maize between countries can be difficult since the importation of plant materials requires permits/inspection to prevent the entry of pests that frequently infest maize. A simple, safe and legal method for shipping maize extracts to the USA was needed to conduct a survey of fumonisins in Central America. The objective was to develop a method for isolating and shipping maize extracts for fumonisin analysis so as to facilitate a survey of fumonisin exposure. The results indicate that fumonisins in acetonitrile:water extracts of maize can be isolated on C18 cartridges, held for at least 3 days at 22°C and then an additional 4 days at 4°C before elution and analysis with no losses. This method allows the importation and analysis of maize samples from foreign locations without complications from international safety concerns.

Influence of Chemical Cues from Host Plants on the Behavior of Neonate <I>Estigmene acrea</I> Larvae (Lepidoptera: Arctiidae)

The role of the chemical cues during the host-finding behavior of neonate Estigmene acrea (Drury) larvae was studied in the laboratory using olfactometer bioassays. Three host plant species of E. acrea were used in the experiments: soybean, Glycine max L. Merr.; tomato, Lycopersicum esculentum Miller; and maize, Zea mays L. The number of larvae showing an attraction toward the leaf volatiles of individual test plants was significantly higher than toward the control air in single-choice olfactometer bioassays. In dual-choice tests, larvae showed a clear preference for the volatiles from soybean over the volatiles from maize and tomato leaves. In contrast, larvae did not show any preference for the maize and tomato volatiles. The locomotory behavior of larvae was clearly affected by hexanic and methanolic soybean extracts. Larvae spent more time walking and walked more distance on the zones containing soybean extracts than on the zones treated with maize and tomato extracts and control solvents. The feeding preference tests revealed that larvae prefer feeding on soybean over maize and tomato foliages.

Attractancy of acetone and diethylether extracts of cereals toOryzaephilus mercator

Acetone and diethylether extracts of maize, millet, sorghum and wheat attracted 1- to 7- and 28- to 42-day-old Oryzaephilus mercatoradults. The 1- to 7-day adults were significantly more responsive than the 28- to 42-day, and adults starved for up to 3 days showed an increasing response. Diethylether extracts were more attractive than acetone extracts and there was a significant interaction between age and solvent. Copyright © 2006 John Wiley & Sons, Ltd

IN VITRO ORGANOGENESIS OF GENTIANA PUNCTATA

Multiplication of Gentiana punctata was obtained from seedlings inoculated into MS medium supplemented with different growth regulators and maize extract. Media supplemented with 2iP or zeatin and IBA ensure a low multiplication. Plant multiplication was slightly improved by addition of maize extract to the culture media. Rhysogenesis was not obtained only on medium supplemented with NAA-1.0 mg/l, 2iP-1.0 mg/l and Me -1.0 mg/l. In vitro plantlets have been transferred to soil after six weeks of culture and acclimatization was successfully obtained.

An analysis of avidin, biotin and their interaction at attomole levels by voltammetric and chromatographic techniques

The electroanalytical determination of avidin in solution, in a carbon paste, and in a transgenic maize extract was performed in acidic medium at a carbon paste electrode (CPE). The oxidative voltammetric signal resulting from the presence of tyrosine and tryptophan in avidin was observed using square-wave voltammetry. The process could be used to determine avidin concentrations up to 3 fM (100 amol in 3 microl drop) in solution, 700 fM (174 fmol in 250 microl solution) in an avidin-modified electrode, and 174 nM in a maize seed extract. In the case of the avidin-modified CPE, several parameters were studied in order to optimize the measurements, such as electrode accumulation time, composition of the avidin-modified CPE, and the elution time of avidin. In addition, the avidin-modified electrode was used to detect biotin in solution (the detection limit was 7.6 pmol in a 6 mul drop) and to detect biotin in a pharmaceutical drug after various solvent extraction procedures. Comparable studies for the detection of biotin were developed using HPLC with diode array detection (HPLC-DAD) and flow injection analysis with electrochemical detection, which allowed biotin to be detected at levels as low as 614 pM and 6.6 nM, respectively. The effects of applied potential, acetonitrile content, and flow rate of the mobile phase on the FIA-ED signal were also studied.

In-gel precipitation of enzymatically released phosphate

The phosphate precipitation reaction using ammonium molybdate and triethylamine under low pH has been applied to gel-based assays for detecting phosphate-releasing enzymes. The sensitivity of the assay is 10 pmol Pi/mm 2 of 1.5-mm-thick gel. The assay is applicable to enzymes with a wide range of optimal pH, from acid (pH 4.5) to alkaline phosphatase (pH 9.7), and to enzymes that use acid-labile substrates such as apyrase and glutamine synthetase. Using a negative staining approach, maltose phosphorylase, a phosphate-consuming enzyme, can also be detected. The assay was used to detect glutamine synthetase isoforms, separated by nondenaturing polyacrylamide gel electrophoresis from crude maize extracts. For downstream applications such as staining gels for proteins, the gels with precipitate should be incubated in 10 mM dithiothreitol or β-mercaptoethanol until the precipitate is dissolved and then thoroughly washed in water. In comparison to calcium phosphate precipitation or the phosphomolybdate–malachite green method, this method is more sensitive. It is a very simple, rapid, versatile, reproducible, and inexpensive method that could be a useful tool in enzymological studies.

Detection of moniliformin in maize using capillary zone electrophoresis

Moniliformin is a mycotoxin produced by certain fungi pathogenic to maize. It is capable of causing disease in domestic animals, possibly through inhibition of pyruvate dehydrogenase. Testing for MON commonly involves extraction of maize, isolation of moniliformin using solid-phase extraction columns and detection with high-performance liquid chromatography (HPLC) or gas chromatography. A capillary zone electrophoresis-diode array detection (CZE-DAD) method for determination of moniliformin in maize is reported. The extraction and isolation procedures are similar to those of a commonly used HPLC method, while the detection step requires only 10 min. Sixty-three samples of maize were tested by an established HPLC method using absorbance at 229 nm (HPLC-ultraviolet light) and by the CZE-DAD method. The limit of detection of the CZE-DAD method was 0.1 μg MON g−1 maize compared with 0.05 μg g−1 for the HPLC-ultraviolet light method. The CZE-DAD method gave good agreement with the HPLC-ultraviolet light method for samples tested at levels up to 1500 μg g−1, with a linear regression of r 2 = 0.996.

Determination of the mycotoxin fumonisin B1 in maize by reversed-phase thin-layer chromatography: a collaborative study

A simple and cost-effective method using thin-layer chromatography for the determination of the mycotoxin fumonisin B1 in maize is described. The analytical method consisted of the extraction of ground maize by shaking with methanol/water (75:25) for 60 min and clean-up of the resultant extract by means of strong anion exchange solid-phase extraction. The purified residue, formed by evaporation of the elution solvent, was reacted with fluorescamine and the fumonisin B1-derivative was separated by reversed-phase thin-layer chromatography using a developing solution of methanol/aqueous 4% potassium chloride (70:30). The derivatized FB1 was readily visualized as a greenish-yellow spot under long wavelength ultraviolet light and quantified by visual comparison with a set of similarly derivatized standards in the range 20–300 ng FB1 spotted on plate. Based on visual comparison, levels down to 0.5 mg kg−1 were successfully estimated. The method was collaboratively studied in 14 laboratories using four duplicate maize meal samples (including a blank) and a spiked sample for determination of recovery. No significant difference was observed between mean FB1 levels by high-performance liquid chromatography or thin-layer chromatography. Based on within-laboratory relative standard deviations of 27.1–41.7% and between-laboratory relative standard deviations of 35.0–63.3%, the method can be considered semiquantitative. The mean recovery achieved by participants at a spiking level of 2.00 mg kg−1 was 74.5%.

PCR-restriction fragment length analysis of aflR gene for differentiation and detection of Aspergillus flavus and Aspergillus parasiticus in maize

Contamination of food and feedstuffs by Aspergillus species and their toxic metabolites is a serious problem as they have adverse effects on human and animal health. Hence, food contamination monitoring is an important activity, which gives information on the level and type of contamination. A PCR-based method of detection of Aspergillus species was developed in spiked samples of sterile maize flour. Gene-specific primers were designed to target aflR gene, and restriction fragment length polymorphism (RFLP) of the PCR product was done to differentiate Aspergillus flavus and Aspergillus parasiticus. Sterile maize flour was inoculated separately with A. flavus and A. parasiticus, each at several spore concentrations. Positive results were obtained only after 12-h incubation in enriched media, with extracts of maize inoculated with A. flavus (10 1 spores/g) and A. parasiticus (10 4 spores/g). PCR products were subjected to restriction endonuclease ( HincII and PvuII) analysis to look for RFLPs. PCR-RFLP patterns obtained with these two enzymes showed enough differences to distinguish A. flavus and A. parasiticus. This approach of differentiating these two species would be simpler, less costly and quicker than conventional sequencing of PCR products.

Relationship between peach lipid transfer protein specific IgE levels and hypersensitivity to non-Rosaceae vegetable foods in patients allergic to lipid transfer protein

Lipid transfer protein (LTP), the major allergen in Rosaceae in geographic areas where the prevalence of birch pollen allergy is low, is a widely cross-reacting pan-allergen, but the pattern of cross-reactivity to plant-derived foods botanically unrelated to Rosaceae shows much variability. To examine the relationship between peach LTP specific IgE levels and cross-reactivity to several non-Rosaceae, plant-derived foods. IgE specific for peach LTP was measured by enzyme-linked immunosorbent assay in serum samples from 40 patients with Rosaceae allergy monosensitized to LTP. Patients were considered monosensitized to this protein in the absence of sensitization to other cross-reacting, plant-derived foods as shown by negative skin prick test (SPT) results with both birch and mugwort pollen. SPTs with commercial extracts of walnut, hazelnut, peanut, celery, maize, rice, tomato, orange, and onion were performed to detect possible immunologic cross-reactivity to these foods. Patients with negative SPT results with non-Rosaceae foods showed significantly lower levels of IgE to peach LTP than patients showing skin reactivity to one or more non-Rosaceae foods (P < .001). A significant difference in specific IgE to peach LTP between patients with positive or negative SPT results was observed with each individual food (P < .001 in all cases). The level of IgE to peach LTP was strongly related to the number of positive SPT results with non-Rosaceae foods (r = 0.78; P < .001). Increasing levels of IgE to peach LTP were associated with skin reactivity to nuts (29/40 [72%]), peanut (27/40 [67%]), maize (16/39 [41%]), rice (14/39 [36%]), onion (13/37 [35%]), orange (9/32 [28%]), celery (11/40 [27%]), and tomato (8/39 [20%]). This study suggests that all allergenic determinants in LTP from vegetable foods other than peach cross-react with peach LTP determinants, whereas only some peach LTP epitopes cross-react with allergenic determinants on botanically unrelated, plant-derived foods. The high levels of IgE to peach LTP seem to reflect the presence of IgE targeting common allergenic determinants of LTP, causing cross-reactivity to botanically unrelated, vegetable foods. In LTP-allergic patients, increasing levels of IgE to peach LTP are paralleled by an increasing number of foods other than Rosaceae positive on SPT that cause clinical symptoms.

XIP-type endoxylanase inhibitors in different cereals

Abstract Highly pure XIP-type (for Xylanase Inhibiting Protein) endoxylanase inhibitor fractions were selectively obtained with a high yield from rye, durum wheat, barley, and maize extracts by affinity chromatography with immobilised Aspergillus niger endoxylanase Xyn1 following removal of the TAXI-type (for Triticum aestivum xylanase inhibitor) endoxylanase inhibitors by affinity chromatography with immobilised Bacillus subtilis endoxylanase XynA. No inhibitors belonging to the XIP family occur in rice, oats, and buckwheat. N-terminal amino acid sequences of the non-wheat XIP-type inhibitors were very similar or identical to those of wheat XIP-1, a chitinase homologue. The isolated inhibitors are basic, monomeric proteins of ca. 30 kDa with pI values of at least 8.5 (rye, durum wheat, and barley XIP) and ca. 7.0 (maize XIP). They are, in general, active against fungal endoxylanases and do not hydrolyse chitin. SDS–PAGE analysis and high-resolution cation exchange chromatography suggest the presence of multiple XIP-type isoinhibitors in the different cereals.

Application of avidin-biotin technology and adsorptive transfer stripping square-wave voltammetry for detection of DNA hybridization and avidin in transgenic avidin maize.

The proteins streptavidin and avidin were electrochemically detected in solution by adsorptive transfer stripping square wave voltammetry (AdTS SWV) at a carbon paste electrode (CPE). AdTS SWV was used to quantify biotinylated oligonucleotides, DNA hybridizations, and avidin in extracts of transgenic avidin maize. The detection limits of denatured and native streptavidin were 6 pM and 120 nM, respectively. The results demonstrated that streptavidin/avidin AdTS SWV is a sensitive and specific method for quantifying DNA and proteins in biological samples such as foods and tissue extracts, including genetically modified crops (avidin maize) and other plants in neighboring fields.

Expression, purification, and initial characterization of a recombinant form of plant PEP-carboxylase kinase from CAM-induced Mesembryanthemum crystallinum with enhanced solubility in Escherichia coli

Plant phospho enolpyruvate-carboxylase kinase (PEPC-kinase [PpcK]) is the smallest Ser/Thr kinase identified to date, having a molecular mass of ∼32,000. This novel, monomeric kinase is dedicated to the phosphorylation of plant PEPC, thereby regulating this target enzyme’s activity and allosteric properties. Although several recombinant, non-fusion PpcK proteins have been produced recently in Escherichia coli, these are plagued by their high degree of insolubility. Here, we report the use of the native, E. coli NusA protein and a related E. coli expression vector (pET-43a(+) [Novagen]) for enhancing the solubility of this recalcitrant Ser/Thr kinase at least 10-fold by its production as a dual 6xHis-tagged NusA/McPpcK1 fusion protein, which accounts for ∼10% of the soluble protein fraction from induced cells. Capture of this fusion protein from the centrifuged cell extract by immobilized metal (Ni 2+) affinity-chromatography, its “on-bead” cleavage by thrombin, and subsequent elution yielded milligram quantities of a “free,” ∼36-kDa form of PpcK for further purification by fast-protein liquid chromatography on blue dextran–agarose or preparative SDS–PAGE. Steady-state kinetic analysis of the former, active preparation revealed that this dedicated kinase discriminates against neither various isoforms of plant PEPC nor certain mutant forms of recombinant C 4 PEPC. Alternatively, the latter, electrophoretically homogeneous sample of the ∼36-kDa polypeptide was used as antigen for polyclonal-antibody production in rabbits. The antibodies against the recombinant McPpcK1 from Mesembryanthemum crystallinum cross-reacted on Western blots with an enriched preparation of the maize-leaf kinase, but not with the parent crude extract, thus directly documenting this protein’s extremely low abundance in vivo. However, these antibodies were effective in immunoprecipitating 32 P -based PpcK activity from crude, desalted extracts of maize leaves and soybean root-nodules.

Type A-trichothecenes — Quantitative analysis using LC-MS and occurrence in Austrian maize and oats

During this study a method was developed for the quantitative determination of diacetoxyscirpenol (DAS), T-2 toxin and HT-2 toxin using deuterated T-2 toxin (T-2 d3 toxin) as an internal standard. The described method involves a clean up step of maize extract by the use of Mycosep® 227 columns a chromatographic separation on a Zorbax® bonus-RP-column (2,1×150mm) and the detection and quantification step on a mass spectrometer in SIM (Selected Ion Monitoring) mode.Data on the occurrence of three type A trichothecenes in Austrian maize, maize silage and oats were collected. It could be shown that maize and silage samples harvested in 2002 were only contaminated to a small extent with T-2 toxin (8% of the maize, 0% of the silage) and with HT-2 toxin (30% of the maize, 18% of the silage). But most of the analysed oats samples showed significant levels of T-2 toxin (64%) or HT-2 toxin (82%).

Sperm extract and inositol 1,4,5-triphosphate induce cytosolic calcium rise in the central cell of Torenia fournieri

Microinjection of soluble sperm extract and Calcium Green-1 10 kDa-dextran conjugate (CG-1) into the mature central cell of Torenia fournieri induced a significant rise in cytosolic free calcium concentration ([Ca2+]i). The rise reached a maximum at 20 min after injection and then steadily declined. Nevertheless, a relatively high level of [Ca2+]i was maintained even 40 min after injection. Microinjection of sperm extract of maize into Torenia central cells, however, did not trigger any increase in [Ca2+]i, suggesting the possibility of distinct triggers in different species. We also injected caged inositol 1,4,5-triphosphate (InsP3) and caged cyclic ADP-ribose (cADPR) into Torenia central cells to compare the pattern of Ca2+ rise induced by the sperm extract. The results showed that [Ca2+]i elevation triggered by the release of InsP3 after photolysis appears much faster than that induced by sperm extract. The increase in [Ca2+]i reached a maximum at 70–80 s and dropped to the resting level within 300 s after photolysis. Microinjection of cADPR, however, did not induce any changes in [Ca2+]i. The results indicate that sperm extract might contain factors triggering the release of Ca2+ in the central cell.

Synthesis of biologically active lipids by fungus Mucor lusitanicus 306D grown on media with various composition

Growth and lipogenesis of fungus Mucor lusitanicus 306 D producing gamma-linolenic acid was studied under various regimes of nitrogen and carbon nutrition. Media containing food industry wastes such as maize extract, molasses, and protein hydrolysate were used. Content of gamma-linolenic acid was higher when using carbohydrates such as glucose and molasses as carbon sources and urea as a nitrogen source. At high glucose concentration (100 g/l), fed batch cultivation provided high content of gamma-linolenic acid in lipids (1 g/l). After extraction of lipids, fungus biomass contained 42% proteins with all essential amino acids. Defatted biomass was shown to be effectively assimilated by minks.

Pengaruh Laju Irigasi Serta Dosis Bahan Pengkondisi Tanah terhadap Tingkat Penahanan Lengas Tanah dan Produksi Tanaman Pangan dan Hortikultura pada Tanah Pasir

Utilization of coastal plain area for farming are scarce due to the problems faced in the development of those lands such as low fertility, high percolation as well as evaporation rate. Temperature ranges from 40-60°C. In order to increase the utilization of the land, an attempt was done to study the effects of number of manure and irrigation rate on the yields and soil water extraction of maize, groundnut, tomato and chilly pepper in sandy soil. A series of experiment w as conducted from April to December 200I using completely randomized design. The treatments included 4 rates of manure i.e. 10, 15,20 and 25 MT/ha and 4 rates of irrigation I.e. 0.6 I/day, 0.9 I/day, 1.21/day and 1.5 I/day. The result of the experiment showed that the favourable condition to keep moisture content in sandy soil at field capacity and flushing of heat in the soil was achieved at irrigation rate of 1.2 I/day except for tomato which obtained the highest water extraction at irrigation rate of 0.9 I/day. Soil water extraction decreased between 0.15 to 0.55% when irrigation rate were reduced from 1.2 I/day to 0.9 I/day and 0.6 I/day on: maize, groundnut and chilly pepper. The highest yield was obtained at fertilization rate of 20 MT/ha except for maize which obtained the highest yield at fertilization rate of I0 MT/ha. Keywords: Coastal plain, Physical properties, Drip irrigation, Organic fertilizer