Development of a sensitive Indirect Competitive Enzyme-Linked Immunosorbent Assay (ic-ELISA) based on the monoclonal antibody for the detection of the imidaclothiz residue

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on monoclonal antibodies (MoAbs) for imidaclothiz was developed. The hapten of imidaclothiz was synthesized and conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to form the artificial antigens. MoAbs were obtained by immunizing BALB/c mice. Under the optimized conditions (10% methanol, 0.14 M Na(+), and pH 7.4), the half-maximal inhibition concentration (IC(50)) was 0.0875 ± 0.0034 mg/L and the limit of detection (IC(20)) was 0.0178 ± 0.0018 mg/L for imidaclothiz. There were no obvious cross-reactivities with most of the structural analogues of neonicotinoid insecticides, except imidacloprid. The recoveries of imidaclothiz in environmental and agricultural samples, including tap water, paddy water, soil, and cabbage, ranged from 80.43 to 113.83%, well within the requirements of residue detection. These results showed that this immunoassay could be used for the determination of imidaclothiz in environmental and agricultural samples.     

Purification and Characterization of a New Class of Insect α-Amylase Inhibitors from Barley

Barley seeds contain proteins that apparently protect them against attack by microorganisms and insects. Studies of these barley defensive proteins may lead to the development of barleys with improved natural resistance to pests. We have purified two low molecular weight proteins, designated BIα1 and BIα2, from barley grain, using ion-exchange chromatography and reversed-phase and gel-permeation high-performance liquid chromatography (HPLC). Both BIα1 and BIα2 inhibited insect (yellow meal worm, Tenebrio molitor) α-amylase activities. For the T. molitor α-amylase, the IC50 values of BIα1 and BIα2 were 80 μg/mL (12.5 μM) and 34 μg/mL (6.8 μM), respectively. Neither protein inhibited either human salivary α-amylase, barley α-amylase, or trypsin activities. N-terminal amino acid sequences of the inhibitors were highly homologous with those of the plant proteins called defensins. The first 20 N-terminal amino acids of BIα2 were identical to those of γ-hordothionin, but neither BIα1 nor BIα2 protein showed any homology with the chloroform-methanol (CM) soluble protein amino acid consensus sequence. The two inhibitors therefore apparently comprise another group of low molecular weight barley proteins that inhibit the α-amylase activities of some insects that attack cereal grains.     

Effects of (1→3)(1→4)-β-d-Glucans of Wheat Flour on Breadmaking

Water-soluble nonstarch polysaccharides were extracted from commercial hard red winter wheat flour and separated into three fractions by graded ethanol precipitation. The three fractions, F15, F40, and F60, varied in polysaccharide composition. Fraction F15 was rich in watersoluble (1→3)(1→4)-β-d -glucans, and fractions F40 and F60 were rich in arabinoxylans. Addition of individual fractions to a bread formula did not affect bread loaf volume. Addition of fraction F15 to the formula improved bread crumb grain. Treatment of (1→3)(1→4)-β-D -glucan-rich fraction F15 with lichenase before its addition to the bread formula resulted in bread with poor crumb grain. Treatment of the F15 fraction with β-xylanase before its addition to the bread formula resulted in bread with slightly improved crumb grain. Presumably, the (1→3)(1→4)-β-D -glucans in fraction F15 improved crumb grain by stabilizing air cells in the bread dough and preventing coalescence of the cells. Addition of pentosan-rich fractions F40 and F60 to the bread formula did not improve crumb grain and interfered with the improving effect of (1→3)(1→4)-β-D -glucan-rich fraction F15. Hydrolysis of the arabinoxylans in flour by adding β-xylanase to the bread formula resulted in improved crumb grain.     

α‐Amylase Inhibitors from Wheat Against Development and Toxigenic Potential of Fusarium verticillioides

Fusarium verticillioides is one of the most important pathogens in maize and is a producer of fumonisin B₁ (FB₁). Although reports of its presence in wheat are scarce, the susceptibility of this cereal to fungus of the same genus motivates interest in investigating compounds present in the grain with inhibitory activity against this species. The aim of this study was to extract α‐amylase inhibitors from wheat and apply them in vitro to evaluate its effect on the development and expression of toxigenic potential of F. verticillioides. The α‐amylase inhibitors, both crude (P₀) and purified (P₁), were applied to in vitro culture containing a pathogen mycelium disc. Mycelial growth of the pathogen, glucosamine content, α‐amylase activity, and production of FB₁ were investigated. All protein extracts of wheat showed the ability to inhibit pathogen growth, especially the extract P₀ from cultivar Quartzo, which resulted in a reduction of glucosamine content (66%) and α‐amylase activity (84%). Furthermore, the protein inhibitors showed antifumonisin effect, reducing by 33 and 47% the mycotoxin production when applied as P₀ and P₁, respectively. These results suggest that α‐amylase inhibitor contributed to resistance against pathogen attack, acting in a diversified manner for each fungal species.     

Low α-Amylase Starch Digestibility of Cooked Sorghum Flours and the Effect of Protein

The comparably low starch digestibility of cooked sorghum flours was studied with reference to normal maize. Four sorghum cultivars that represent different types of endosperm were used. Starch digestibilities of 4% cooked sorghum flour suspensions, measured as reducing sugars liberated following α-amylase digestion, were 15–25% lower than for cooked maize flour, but there were no differences among the cooked pure starches. After the flours were predigested with pepsin to remove some proteins, the starch digestibility of cooked sorghum flours increased 7–14%, while there was only 2% increase in normal maize; however, there was no effect of pepsin treatment on starch digestibility if the flours were first cooked and then digested. After cooking with reducing agent, 100 mM sodium metabisulfite, starch digestibility of sorghum flours increased significantly while no significant effect was observed for maize. Also, starch solubility of sorghum flours at 85 and 100°C was lower than in maize, and sodium metabisulfite increased solubility much more in sorghum than in maize. Differential scanning calorimetry results of the flour residue after α-amylase digestion did not show any peaks over a temperature range of 20–120°C, indicating that sorghum starches had all undergone gelatinization. These findings indicate that the protein in cooked sorghum flour pastes plays an important role in making a slowly digesting starch.     

Large-Scale Purification and Characterization of Barley Limit Dextrinase,a Member of the α-Amylase Structural Family

Homogeneous barley limit dextrinase (LD) was isolated on a large scale in a yield of 9 mg/kg of 10-day germinated green malt. This represents a 9,400-fold purification and 29% recovery of the activity in a flour extract in 0.2M NaOAc (pH 5.0) containing 5 mM ascorbic acid. The purification protocol consists of precipitation from the extract at 20–70% saturated ammonium sulfate (AMS), followed by diethylaminoethyl (DEAE) 650S Fractogel anion-exchange chromatography, and affinity chromatography on β-cyclodextrin-Sepharose in the presence of 2M AMS. LD was eluted by 7 mMβ-cyclodextrin and contains a single polypeptide chain of 105 kDa (SDS-PAGE) and pI 4.3. Sequence analysis of tryptic fragments, prepared from 2-vinylpyridinylated LD and purified by RP-HPLC, identified short motifs recognized in β-strand 2, 3, and 5 characteristic of a catalytic (β/α)₈-barrel domain of the α-amylase family of amylolytic enzymes. Barley LD has ≈50 and 85% sequence identity to bacterial pullulanases and rice starch debranching enzyme, respectively. By using ¹H-NMR spectroscopy, LD hydrolyzes specifically α-1,6-glucosidic linkages in pullulan and a branched oligodextrin, 6²-O-α-maltotriosyl-maltotriose, with retention of the α-anomeric configuration. β-Cyclodextrin competitively inhibits the LD activity with K_i of 40 μM, while K_i is 1.9 mM and 2.4 mM for α-cyclodextrin and γ-cyclodextrin, respectively.     

Detection,Localization, and Variability of Endogenous β‐Glucanase in Wheat Kernels

Clinical studies with isolates of β‐glucan have shown that the health benefits are regulated not only by the polysaccharide concentration but also by the molecular weight and concentration in solution, because these health benefits are controlled, inter alia, by viscosity in the gut. The degradation of β‐glucan in baked products is likely caused by baking ingredients or processes, or by endogenous enzymes in wheat flour. The objectives of the present study were to quantify β‐glucanase in wheat kernels and to determine factors that influence the levels of this enzyme. A modified protocol to quantify β‐glucanase was developed and then confirmed through high‐performance size‐exclusion chromatography (HPSEC) with Calcofluor detection. Under this protocol, it was shown that the concentration of β‐glucanase activity was the highest in the bran fraction of the kernel in ungerminated wheats, whereas it was distributed throughout the entire kernel following germination. Furthermore, investigation on different wheat cultivars planted in the same and different locations showed that genotype, environment, and agronomic practice all can have an effect on β‐glucanase activity level in wheat kernels.     

Mixed Linkage (1→3),(1→4)‐β‐d‐Glucans of Grasses

The mixed‐linkage (1→3),(1→4)‐β‐d ‐glucans are unique to the Poales, the taxonomic order that includes the cereal grasses. (1→3), (1→4)‐β‐Glucans are the principal molecules associated with cellulose microfibrils during cell growth, and they are enzymatically hydrolyzed to a large extent once growth has ceased. They appear again during the developmental of the endosperm cell wall and maternal tissues surrounding them. The roles of (1→3),(1→4)‐β‐glucans in cell wall architecture and in cell growth are beginning to be understood. From biochemical experiments with active synthases in isolated Golgi membranes, the biochemical features and topology of synthesis are found to more closely parallel those of cellulose than those of all other noncellulosic β‐linked polysaccharides. The genes that encode part of the (1→3),(1→4)‐β‐glucan synthases are likely to be among those of the CESA/CSL gene superfamily, but a distinct glycosyl transferase also appears to be integral in the synthetic machinery. Several genes involved in the hydrolysis of (1→3),(1→4)‐β‐glucan have been cloned and sequenced, and the pattern of expression is starting to unveil their function in mobilization of β‐glucan reserve material and in cell growth.     

Structure of (1→3)(1→4)‐β‐d‐Glucan in Waxy and Nonwaxy Barley

The endosperm cell walls of barley are composed largely of a (1→3)(1→4)‐β‐d ‐glucan commonly known simply as β‐d ‐glucan (Wood 2001). There has been much research into the characteristics of barley β‐glucan because of the influence of this polysaccharide on performance of barley in malting and subsequent brewing of beer, and in feed value, especially for young chicks (MacGregor and Fincher 1993). The potential for β‐glucan to develop high viscosity is a problem in these uses, but from the perspective of human nutrition, this characteristic may be an advantage. The glycemic response to oat β‐glucan is inversely related to (log)viscosity (Wood et al 1994a) and there is evidence to suggest that the lowering of serum cholesterol levels associated with oat and barley products (Lupton et al 1994; Wood and Beer 1998) is at least in part due to the β‐glucan (Braaten et al 1994) and probably also its capacity to develop viscosity in the gastrointestinal tract (Haskell et al 1992).     

Effects of Mutant Thermostable α‐Amylases on Rheological Properties of Wheat Dough and Bread

Thermostable mutant α‐amylases (21B, M111, and M77) with various degrees of thermostability were purified from Bacillus amyloliquefaciens F and used as improvers for breadmaking. Test baking with the mutant enzymes was conducted using the long fermentation sponge‐dough method. Addition of an appropriate amount of mutant α‐amylases to the ingredients distinctly increased the specific volume of the bread and improved the softness of breadcrumb as compared with the addition of Novamyl (NM), an exo‐type α‐amylase. M77 was the most effective in retarding the staleness of breadcrumb. The softness of breadcrumb during storage, however, was not correlated with the thermostability. All mutant α‐amylases weakened the mixing property of the dough, whereas they strengthened the property of fermented dough. Especially, M77 and NM had different effects on the dough properties, but their bread qualities were similar to each other. The strong tolerance of M77 dough to the long baking process might be due to the production of hydrolyzed starches, oligosaccharides in the range of maltopentaose to maltohexaose, as compared with NM. Therefore, in the light of present findings, these mutant α‐amylases are possible substitutes for NM as bread improvers.     

Deduced Amino Acid Sequence of an α-Gliadin Gene from Spelt Wheat (Spelta) Includes Sequences Active in Celiac Disease

The complete amino acid sequence of an α-type gliadin from spelt wheat (spelta) has been deduced from the cloned DNA sequence and compared with α-type gliadin sequences from bread wheat. The comparison showed only minor differences in amino acid sequences between the α-type gliadin from bread wheat and the α-type gliadin from spelta. The two sequences had an identity of 98.5%. Larger differences can be found between different α-type gliadin amino acid sequences from common bread wheat. Because all the different classes of gliadins, α, β, γ, and ω, appear to be active in celiac disease, it is reasonably certain that the spelta gliadin is also toxic. We conclude that spelta is not a safe grain for people with celiac disease, contrary to the implications in labeling a bread made from spelta as “an alternative to wheat”. Our conclusions are in accord with spelta and bread wheat being classed taxonomically as subspecies of the same genus and species, Triticum aestivum L.     

Effects of Pearling on Falling Number and α‐Amylase Activity of Preharvest Sprouted Spring Wheat

Preharvest sprouted wheat is often characterized by the falling number (FN) test. FN decreases in preharvest sprouted wheat as enzymatic degradation of the starchy endosperm increases. Wheat with FN values <250–275 is often discounted at the time of sale. The intent of this investigation was to evaluate the effects of debranning or pearling on the flour quality traits of five samples of wheat rated as low, med‐low, medium, med‐high, and sound that exhibited a range in FN values of 62–425 sec. Replicates of each sample were pearled for 30, 60, and 120 sec to remove portions of the outer bran layers before milling. FN was highly correlated with α‐amylase activity (r > ‐0.97) in the med‐low, medium, and med‐high FN sample sets as pearling time increased. FN increased in the medlow, medium, and med‐high FN samples by 128, 123, and 80%, respectively, after 120 sec of pearling. Pearling had no effect on flour FN of the low FN sample but α‐amylase activity was significantly decreased. Pearling had little or no effect on FN and α‐amylase activity of the sound sample. FN was moderately to strongly correlated with Rapid Visco Analyser (RVA), alveograph, and farinograph properties, and poorly correlated with protein content, flour yield, and bread loaf volume. In subsequent breadmaking studies, bread loaf volume, and crumb characteristics of flour from pearled wheat were not significantly different from loaf volume and crumb characteristics of flour from the corresponding nonpearled wheat.     

Development of a sensitive ELISA for the determination of fumonisin B(1) in cereals

Monoclonal fumonisin B(1) antibodies with high titer were raised by using FB(1)-glutaraldehyde-keyhole limpet hemocyanin immunogen prepared by a short cross-linker reagent (glutaraldehyde). Mean cross-reactivities of the selected monoclonal antibody for FB(1), FB(2), and FB(3) were 100, 91.8, and 209%, respectively; no reactivity was found with hydrolyzed fumonisin. A direct, competitive enzyme-linked immunosorbent assay for the quantitative determination of FB(1) in cereals has been developed with this antibody. Fifty percent acetonitrile-based solvent with some additives was used for extraction of cereals, and the diluted extracts were used without cleanup in the test. The mean within-assay and interassay coefficients of variation for the standard curve were <10%. The measuring range of this test is 10-500 ng/g, with a detection limit of 7.6 ng/g FB(1). The toxin recovery from cereals infected with 50-200 ng/g of FB(1) varied between 61 and 84%. According to the comparable results of naturally infected maize samples, this test proved to be suitable for the rapid screening of food and feed samples for the presence of FBs.     

Two‐Site Sandwich ELISA for Discriminating Different Gli‐1 (gliadin)/Glu‐3 (LMW‐glutenin subunit) Alleles in Hexaploid Wheat

A panel of monoclonal antibodies was assessed in a two‐site sandwich ELISA format, using both reduced glutenin subunit and gliadin‐rich antigen preparations, to develop assays that could potentially discriminate between Gli‐1/Glu‐3 allelic variants in hexaploid wheat. Each antibody was assessed as the immobilized and the enzyme‐labeled antibody in the sandwich ELISA. A number of antibody combination were identified which could discriminate different Gli‐1/Glu‐3 allelic variants in a population of doubled haploid lines derived from a cross between parents that differed at each of these loci. Certain labeled antibodies consistently detected allelic variation at a particular locus when used in conjunction with any of several immobilized antibodies. However, the level of discrimination was largely dependent on the choice of immobilized antibody. Two antibody combinations were identified that provided twofold differences in ELISA absorbances in flour extracts from different allelic variants at the Gli‐A1/Glu‐A3 and Gli‐B1/Glu‐B3 loci. By analyzing the prolamin composition of the antigen preparations, and the performance of the assays with flour extracts from a set of Gli‐1/Glu‐3 biotypes and a range of diverse cultivars, the biochemical basis for the discrimination was determined. The assays may have potential for use in high‐throughput screening in wheat breeding programs.     

Development of New Method for Extraction of α‐Zein from Corn Gluten Meal Using Different Solvents

A modified procedure for the extraction of α‐zein from corn gluten meal was developed and compared against a commercial extraction method. The modification involved raising the concentration of alcohol in solvent and removing the precipitate by centrifugation. Five organic solvent mixtures were compared using the modified extraction procedure developed along with the reductant sodium bisulfite and NaOH. The modified procedure precipitated most of the non‐α‐zein protein solids by increasing the concentration of alcohol. The supernatant had α‐zein‐rich fraction, resulting in higher yield of α‐zein than the commercial method when cold precipitated. The commercial extraction procedure had a zein yield of 23% and protein purity of 28% using 88% 2‐propanol solvent. The three best solvents, 70% 2‐propanol, 55% 2‐propanol, and 70% ethanol, yielded ≈35% of zein at protein purity of 44% using the modified extraction procedure. Zeins extracted using the novel method were lighter in color than the commercial method. Densitometry scans of SDS‐PAGE of α‐zein‐rich solids showed relatively large quantities of α‐zein with apparent molecular weights of 19,000 and 22,000 Da. The α‐zein‐rich solids also had small amounts of δ‐zein (10,000 Da) because it shares similar solubility properties to α‐zein. A solvent mixture with 70% 2‐propanol, 22.5% glycerol, and 7.5% water extracted significantly less zein (≈33%) compared to all other solvents and had α‐zein bands that differed in appearance and contained little to no δ‐zein.     

Adsorption of glycerophosphate on goethite (α‐FeOOH): A macroscopic and infrared spectroscopic study

Adsorption, desorption, and precipitation reactions at environmental interfaces govern the bioavailability, mobility, and fate of organic phosphates in terrestrial and aquatic environments. Glycerophosphate (GP) is a common environmental organic phosphate, however, surface adsorption reactions of GP on soil minerals have not been well understood. The adsorption characteristics of GP on goethite were studied using batch adsorption experiments, zeta (ζ) potential measurements, and in situ attenuated total reflectance‐Fourier transform infrared spectroscopy (ATR‐FTIR). GP exhibited fast initial adsorption kinetics on goethite, followed by a slow adsorption. The maximum adsorption densities of GP on goethite were 2.00, 1.95, and 1.44 μmol m^?2 at pH 3, 5, and 7, respectively. Batch experiments showed decreased adsorption of GP with increasing pH from 3 to 10. Zeta potential measurements showed a remarkable decrease in the goethite isoelectric point upon GP adsorption (from 9.2 to 5.5), suggesting the formation of inner‐sphere surface complexes. In addition, the ATR‐FTIR spectra of GP sorbed on goethite were different from those of free GP at various pH values. These results suggested that GP was bound to goethite through the phosphate group by forming inner‐sphere surface complexes.