Comparative efficiencies of isopropyl and tert-butyl alcohols for extracting zeins from maize endosperm

Protein of endosperm of maize grains originating from three wild-type inbreds and their opaque-2 versions were solubilized in diverse extracts (E) by the sequential use of 0.5 M NaCl, water (E(1,2)), alcohol plus a reducing agent (E(3)), and salt plus a reducing agent (E(4)). Zeins were isolated in extracts E(3) and E(4) obtained by using 55% (w/w) isopropyl alcohol (i-PrOH) + 0.2% dithiothreitol (DTT) followed by 0.5 M NaCl + 0.2% DTT buffered at pH 10 or 60% tert-butyl alcohol (t-BuOH) + 0.2% DTT followed by 0.5% sodium acetate + 0.2% DTT in 30% t-BuOH. For a given genotype the percentage of extracted zeins was independent of the nature of the alcohol. The latter had a slight effect on the respective magnitude of E(3) and E(4): E(3) increased at the expense of E(4) when t-BuOH was substituted to i-PrOH for their isolation. The percentage of the total endosperm nitrogen present in E(3) + E(4) was identical to that of fractions F(II) + F(III) + F(IV) isolated according to the classical Landry-Moureaux extraction procedure. SDS-PAGE analysis revealed the presence of all types of zeins (alpha, beta, gamma, and delta) in E(3) and F(III), residual zeins in E(4) isolated with t-BuOH, and streaking only in E(4) and F(IV) isolated with NaCl at pH 10. The data together with those of the literature were discussed with regard to the influence of procedure on the yield of zeins using alcoholic extraction.     

Isolation of tetra-acyl sucrose esters from turkish tobacco using supercritical fluid CO2 and comparison with conventional solvent extraction

Pure supercritical CO2 at various pressures and temperatures was used to effect the fractionation of tetra-acyl sucrose esters (SE) from dried, ground Turkish tobacco without any further pretreatment of the matrix. It was determined that SE cannot be extracted using low density CO2 (150 atm, 60 degrees C, and 0.62 gm/mL or 200 atm, 100 degrees C, and 0.49 gm/mL), whereas other analytes, which strongly interfere with the conventional solvent extraction of SE, can be easily removed under the same conditions. At the higher temperature (100 degrees C), these same analytes that interfere with the conventional solvent extraction of SE are even more readily removed, while the very poor extractability of SE is not affected. It was demonstrated, however, that SE can be removed from the pre-extracted tobacco with supercritical CO2 if the density is greater than (or equal to) 0.73 gm/mL. The supercritical fluid extraction method has been compared with other previous extraction methods that employ conventional solvents. This study provides one of the clearest examples of how the variable density property of a supercritical fluid can be utilized to effect the fractionation of a complex mixture.     

Dichloromethane as a solvent for lipid extraction and assessment of lipid classes and fatty acids from samples of different natures

The usefulness of the solvent mixture dichloromethane/methanol for lipid extraction and the determination of lipid classes and fatty acids in samples of different natures was conducted. Two different extraction methods were compared, one containing chloroform/methanol, another containing dichloromethane/methanol. Total lipid extraction showed some minor differences but no variation in the lipid classes. Regarding the fatty acid profile, in Echium virescens seeds, 17 major fatty acids could be identified and quantified, and all were equally extracted when either solvent system was employed. In Echium acanthocarpum hairy roots, 17 major fatty acids were quantified, showing some statistical differences for one cell line in favor of chloroform. The data obtained from the liquid nutrient medium were also comparable. The cod roe sample showed 31 major fatty acids, showing no statistical differences between the two solvent systems. Contrarily, the CH 2Cl 2 method was able to extract 31 main fatty acids found in European seabass dorsal muscle more efficiently than the CHCl 3 method. The results indicate that, for lipid extraction and fatty acid assessment, dichloromethane/methanol can readily replace the commonly employed chloroform/methanol, thus avoiding the major health, security, and regulatory problems associated with the use of chloroform.     

Effects of extraction solvent mixtures on antioxidant activity evaluation and their extraction capacity and selectivity for free phenolic compounds in barley (Hordeum vulgare L.)

Four kinds of solvent extracts from three Chinese barley varieties (Ken-3, KA4B, and Gan-3) were used to examine the effects of extraction solvent mixtures on antioxidant activity evaluation and their extraction capacity and selectivity for free phenolic compounds in barley through free radical scavenging activity, reducing power and metal chelating activity, and individual and total phenolic contents. Results showed that extraction solvent mixtures had significant impacts on antioxidant activity estimation, as well as different extraction capacity and selectivity for free phenolic compounds in barley. The highest DPPH* and ABTS*+ scavenging activities and reducing power were found in 80% acetone extracts, whereas the strongest *OH scavenging activity, O2*- scavenging activity, and metal chelating activity were found in 80% ethanol, 80% methanol, and water extracts, respectively. Additionally, 80% acetone showed the highest extraction capacity for (+)-catechin and ferulic, caffeic, vanillic, and p-coumaric acids, 80% methanol for (-)-epicatechin and syringic acid, and water for protocatechuic and gallic acids. Furthermore, correlations analysis revealed that TPC, reducing power, DPPH* and ABTS*+ scavenging activities were well positively correlated with each other (p < 0.01). Thus, for routine screening of barley varieties with higher antioxidant activity, 80% acetone was recommended to extract free phenolic compounds from barley. DPPH* scavenging activity and ABTS*+ scavenging activity or reducing power could be used to assess barley antioxidant activity.     

Multiresidue method for the analysis of pesticide residues in fruits and vegetables by accelerated solvent extraction and capillary gas chromatography

An analytical procedure using accelerated solvent extraction and capillary gas chromatography with electron capture and flame photometric detections was developed to simultaneously determine residues of different pesticides in fruits and vegetables. Single laboratory validation of the method was carried out for 28 compounds selected from eight pesticide classes, in blank and fortified samples of fresh pear, cantaloupe, white potato, and cabbage. The method had to meet specific established validation criteria for regulatory purposes applicable to our laboratory. At each of the two fortification levels studied, 24 of the 28 pesticides gave recoveries of more than 70% with a coefficient of variation of less than 10%. With respect to existing procedures, the method showed acceptable limits of detection (from 0.0019 to 0.14 microg/g depending on the pesticide and matrix) while minimizing environmental concerns, time, and labor.     

In search of a maize ideotype for cell wall enzymatic degradability using histological and biochemical lignin characterization

Grass cell wall degradability is conventionally related to the lignin content and to the ferulic-mediated cross-linking of lignins to polysaccharides. To better understand the variations in degradability, 22 maize inbred lines were subjected to image analyses of Fasga- and M?ule-stained stem sections and to chemical analyses of lignins and p-hydroxycinnamic acids. For the first time, the nearness of biochemical and histological estimates of lignin levels was established. Combination of histological and biochemical traits could explain 89% of the variations for cell wall degradability and define a maize ideotype for cell wall degradability. In addition to a reduced lignin level, such an ideotype would contain lignins richer in syringyl than in guaiacyl units and preferentially localized in the cortical region rather than in the pith. Such enrichment in syringyl units would favor wall degradability in grasses, contrary to dicots, and could be related to the fact that grass syringyl units are noticeably p-coumaroylated. This might affect the interaction capabilities of lignins and polysaccharides.     

Establishment of a core collection for maize germplasm preserved in Chinese National Genebank using geographic distribution and characterization data

Since the 1980s, a large collection of maize has been preserved in the China National Genebank. To facilitate the evaluation and utilization of these valuable germplasm resources, a core collection was established. The collection was firstly divided into two groups, i.e. the landraces and inbred lines. The percentage of the original collection to be included in the core was given as 7% based on a previous study of sampling strategies in maize. Each group was sequentially stratified based on administrative provinces or regions and kernel types, and a clustering method was applied in further stratification. A logarithmic strategy was used to determine the number of entries in the core at each step. The process resulted in the maize core comprising 951 landraces and 242 inbred lines. Shannon-Weaver diversity index and means were used to validate the core. The core can be used effectively in further in-depth research and maize improvement.     

SFE-plus-C(18) lipid cleanup and selective extraction method for GC/MS quantitation of polycyclic aromatic hydrocarbons in smoked meat

In biological matrixes lipid material often poses an interference problem for determinations of nonpolar compounds, e.g., polycyclic aromatic hydrocarbons (PAHs). A newly developed supercritical fluid extraction plus adsorbent method, "SFE-plus-C(18)", offers selective extraction of PAHs in lipid-rich biological matrixes without the need for supplementary cleanup. This method eliminates the use of large volumes of toxic solvent and lengthy lipid removal procedures. This study reports the first application of the SFE-plus-C(18) method to the analysis of a genuine food product, i.e., smoked meat (beef). The procedure employs the addition of C(18) adsorbent beads to the initial sample slurry of pureed smoked meat prior to supercritical CO(2) extraction and GC/MS quantitation. During SF extraction, indigenous lipids are preferentially retained on the beads, and PAHs are selectively extracted with supercritical CO(2). In a comparison of determinations of PAHs by SFE-plus-C(18) vs the conventional SFE method, only 11-17% of the indigenous lipids observed by the conventional SFE method were co-extracted using the SFE-plus-C(18) method. The PAHs in smoked meat could thus be determined efficiently in the presence of a reduced background of co-extracted lipids. Out of 10 targeted PAHs, seven were detected with a range of 10.0-26.0 ng/g in the smoked meat sample. The other three PAHs were not present above the detection limit of the instrument (2.5-4.1 pg). The recoveries of PAHs obtained using the conventional SFE method were 63-94% lower than those achieved by SFE-plus-C(18).     

Determination of folate concentrations in diverse potato germplasm using a trienzyme extraction and a microbiological assay

Folate deficiency is a leading cause of birth defects and is implicated in several other diseases. We are interested in how much folate concentrations vary among potato germplasm. We determined total folate concentrations of potato tubers from 67 cultivars, advanced breeding lines, or wild species. Folates were extracted by a tri-enzyme treatment and analyzed by using a Lactobacillus rhamnosus microbiological assay. Folate concentrations varied from 521 +/- 96 to 1373 +/- 230 ng/g dry weight and were genotype and location dependent. The highest folate concentrations were mostly found in color-fleshed potatoes. Variations of folate concentrations within either color- or white-fleshed tubers were similar ( approximately 2-fold). Skin contained approximately 30% higher folate concentrations than flesh. Storage of tubers for 7 months generally led to an increase in folate contents. Semiquantitative RT-PCR analyses showed that higher folate contents were correlated with lower mRNA expression of some folate genes.     

Development of a solid-phase extraction method for phenoxy acids and bentazone in water and comparison to a liquid-liquid extraction method

A rapid solid-phase extraction (SPE) method was developed for the determination of bentazone and the phenoxy acids 2,4-D, dichlorprop, MCPA, and mecoprop in Norwegian environmental water samples. Cartridges with a high-capacity cross-linked polystyrene-based polymer were used for off-line preconcentration. The effects of elution solvent, elution volume, sample volume, sorbent mass, pH, and flow rate on the recoveries of the pesticides were investigated using HPLC. Average recovery of >90% was achieved with 500 mg sorbents using 2 mL of methanol with 5% NH3 as elution solvent. The recoveries were independent of sample pH in the tested range of pH 1-7. Using a sample volume of 200 mL, the limits of determination for the phenoxy acids and bentazone are 0.02 microg/L. Sample volumes up to 2000 mL at a flow rate of 60 mL/min could be handled without any loss of analytes, which makes it possible to lower the limits of determination. The SPE method was compared to a routinely used liquid-liquid extraction method. Three different water matrices spiked at 1.0 and 0.05 microg/L were extracted, and the quantification was performed by GC-MS. Both methods permitted the determination of phenoxy acids and bentazone in distilled water, creek water, and well water down to a level of 0.05 microg/L with recoveries >80% for 200 mL samples. Important advantages of the SPE method compared to the liquid-liquid extraction method were the short extraction times, lack of emulsions, use of disposable equipment, and reduced consumption of organic solvents.     

Determination of organochlorine and organophosphorus pesticide residues in eggs using a solid phase extraction cleanup

A multiresidue solid phase extraction (SPE) method for the isolation and subsequent gas chromatographic determination of nonpolar organochlorine and polar organophosphorus pesticide residues in eggs is described. The method uses an acetonitrile extraction followed by an SPE cleanup using graphitized carbon black and aminopropyl SPE columns. Organophosphorus pesticides are determined by gas chromatography with flame photometric detection. After further cleanup of the extract using Florisil SPE columns, organochlorine pesticides are determined by gas chromatography with electron capture detection. Studies were performed using eggs containing both fortified and incurred pesticide residues. The average recoveries were 86-108% for 8 fortified organochlorine pesticide residues and 61-149% for 28 fortified organophosphorus pesticide residues.     

Comparison of stirring and vortex mixing for the extraction of available nutrients from soil using a laboratory robot

Abstract

We investigated the efficiency with which a laboratory robot is able to extract available nutrients from soil contained in tubes. We have shown that for vortex mixing, the volume of soil and extractant are of critical importance. In cases where the soil/extractant volumes are small (e.g. 2 mL soil: 10 mL extractant), extraction of all the nutrients investigated was good. However, keeping the ratio the same, but increasing the volumes to 6 mL soil:30 mL extractant leads to an overall decrease in extracting efficiency. Using a paddle stirrer to mix the soil and extractant together results in the same extraction efficiency across the range of soil/ extractant volumes.     

Optimization of a solid-phase extraction method using centrifugation for the determination of 16 polycyclic aromatic hydrocarbons in water

A fast and reliable method for the determination of polycyclic aromatic hydrocarbons (PAHs) in water samples by solid-phase extraction (SPE) using centrifugation has been developed and optimized. A silica-based C18 cartridge was used; parameters affecting the extraction procedure such as type and volume of the elution solvent, breakthrough volume of the percolated water sample, drying of the sorbent, and evaporation of the elute have been studied. The innovation of this work was the examination of the use of a centrifugation technique in both the drying and elution steps. When combined with centrifugation, the volume of the elution solvent was reduced to 1 mL and the time for sorbent drying decreased also to 10 min under vacuum. Under optimal conditions, recoveries for the 16 U.S. EPA PAHs were between 70 and 85% and the relative standard deviation varied between 1 and 14%. Surrogate standard recoveries were similarly between 61 and 94% with a relative standard deviation between 2 and 15%. The simplicity of the described method, use of less of organic solvent, short procedure time, and good recoveries demonstrate the advantages of this environmentally friendly approach for routine analysis of numerous samples.     

On-line supercritical fluid extraction/enzymatic hydrolysis of vitamin a esters: a new simplified approach for the determination of vitamins a and e in food

An on-line supercritical fluid extraction (SFE)/enzymatic hydrolysis procedure using immobilized lipase has been developed for the determination of vitamin A in dairy and meat products. Several lipases were tried, of which Novozyme 435 (Candida antarctica type B) showed the highest activity toward retinyl palmitate. There was no observed activity with alpha-tocopheryl acetate. When pressure, temperature, modifiers, flow rate, extraction time, and water content were varied, high vitamin A recovery was obtained in milk powder. Collected extracts were analyzed by reversed-phase high-performance liquid chromatography with ultraviolet and fluorescence detection without additional sample cleanup. The procedure gave reliable values of vitamin A as well as of vitamin E in other food items such as infant formula, minced pork and beef meat, and low- and high-fat liver paste. The described method is faster and more automated than conventional methods based on liquid-liquid extraction, or SFE using off-line saponification, for vitamin A and E determination. Results obtained with the new method did not differ significantly from those obtained with the other two methods mentioned above.     

Allelic diversity of a beer haze active protein gene in cultivated and Tibetan wild barley and development of allelic specific markers

The formation of haze is a serious quality problem in beer production. It has been shown that the use of silica elute (SE)-ve malt (absence of molecular weight (MW) ～14000 Da) for brewing can improve haze stability in the resultant beer, and the protein was identified as a barley trypsin inhibitor of the chloroform/methanol type (BTI-CMe). The objectives of this study were to determine (1) the allelic diversity of the gene controlling BTI-CMe in cultivated and Tibetan wild barley and (2) allele-specific (AS) markers for screening SE protein type. A survey of 172 Tibetan annual wild barley accessions and 71 cultivated barley genotypes was conducted, and 104 wild accessions and 35 cultivated genotypes were identified as SE+ve and 68 wild accessions and 36 cultivated genotypes as SE-ve. The allelic diversity of the gene controlling BTI-CMe was investigated by cloning, alignment, and association analysis. It was found that there were significant differences between the SE+ve and SE-ve types in single-nucleotide polymorphisms at 234 (SNP(234)), SNP(313), and SNP(385.) Furthermore, two sets of AS markers were developed to screen SE protein type based on SNP(313). AS-PCR had results very similar to those obtained by immunoblot method. Mapping analysis showed that the gene controlling the MW～14 kDa band was located on the short arm of chromosome 3H, at the position of marker BPB-0527 (33.302 cM) in the Franklin/Yerong DH population.     

Identification of active bacteria involved in decomposition of complex maize and soybean residues in a tropical Vertisol using N-DNA stable isotope probing

As limited information is available about the relationship between microbial processes and community structure in tropical soils, we used ¹⁵N-DNA stable isotope probing (¹⁵N-DNA-SIP) to identify bacteria actively involved in decomposition of plant residues of different biochemical quality. ¹⁵N-labeled (90 atom%) and unlabeled (control) maize (C-to-N ratio: 32; cellulose content: 24.9%) and soybean (15; 15.5%) leaf residues were incubated in a tropical Vertisol for 15 days. Soil DNA was isolated, subjected to ¹⁵N-DNA-SIP and buoyant density-resolved DNA fractions were analyzed by 16S rRNA gene-based denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of selected DGGE bands. Residue addition induced new bands and changed relative intensity of already existing bands in ¹⁵N-enriched SIP fractions. Phylogenetic analysis of selected, cloned DGGE bands from ‘heaviest’ ¹⁵N-enriched fractions (57.8 atom% (maize), 87.1 atom% (soybean)) revealed that soils treated with maize residues were dominated by Pseudonocardia sp., while Arthrobacter sp. and Streptomyces sp. were found in the soybean residue treated soils. Sequences related to Bacillus sp. and Saccharopolyspora sp. were found in both organic residue treatments. Our study gave clear evidence that ¹⁵N-DNA-SIP combined with 16S rRNA gene-based community fingerprinting of density-resolved fractions and an unlabeled control was suited for detecting active bacteria involved in decomposition of complex maize and soybean residues. In conclusion, we could show that residue quality, inducing contrasting N assimilation by decomposing bacteria, was a substantial determinant of certain decomposing community members assayed in this study.     

Optimization and validation of analytical conditions for cholesterol and cholesterol oxides extraction in chicken meat using response surface methodology

The analytical conditions for the extraction of cholesterol and cholesterol oxides in chicken meat were optimized by means of response surface methodology. The separation and identification were performed by normal phase HPLC using UV and refractive index (RI) detectors, and the confirmation of the 11 cholesterol oxides identities in the samples was verified by HPLC-APCI-MS. The developed methodology showed good analytical performance, presenting recovery levels from 84 to 103% and detection limits varying from 0.01 to 0.06 microg/g for UV detection and from 1.98 to 2.12 microg/g for RI detection. The present study demonstrated the presence of 22 R-hydroxycholesterol, 24 S-hydroxycholesterol, and 22 S-hydroxycholesterol for the first time in chicken meat.     

Determination of calcium,magnesium, zinc and manganese in plant tissue using a dilute HCl extraction method1

Abstract

A non digestion method described earlier for K determination was tested for its ability to extract several other elements in plant tissues of sorghum, pearl millet, chickpea, and pigeonpea. The method involves shaking 0.5 g finely ground (<0.4 mm) plant sample with 40 ml of 0.5 N HCl for 5 minutes at room temperature (25°C) followed by measurement of element concentrations in the filtrate. The values of Ca, Mg, Zn, and Mn obtained by this method were in good agreement with those obtained using the triacid digestion technique. However, this was not the case for P, Fe and Cu. The precision obtained in determining Ca, Mg, Zn and Mn by the HCl extraction method was generally comparable to that obtained by the triacid digestion method. These results suggest that the HCl extraction method can be used for routine and rapid analysis of plant tissues for Ca, Mg, Zn and Mn contents as well as for K content.     

Arcelin in wild bean (Phaseolus vulgaris L.) seeds: sequence of arcelin 6 shows it is a member of the arcelins 1 and 2 subfamily

Arcelins are lectin-related proteins detected only in wild beans collected in Mexico and their presence has been related to resistance against the bean weevils. Six arcelin variants have been described and the gene sequence of four of them was determined. We have isolated and sequenced a cDNA clone encoding arcelin 6. Sequence data indicated that this protein is closely related to arcelin 1 and arcelin 2. The cluster dendrogram produced with the multiple alignment of the cDNA clones coding for arcelins showed that arcelins can be divided into three subgroups: i) arcelin 1, arcelin 2 and arcelin 6, ii) arcelin 4 and iii) arcelin 5a and arcelin 5b. Biochemical data indicate that arcelin 3 belongs to the same subgroup of arcelin 4. Southern blot analysis of genomic DNA yielded similar restriction patterns among members of each subgroup, confirming the results obtained with the multiple alignment. Furthermore, the hybridisation patterns were specific for each arcelin variant. On the basis of these evidences, we suggest that the lectin locus could be a useful tool for understanding evolutionary relationships in common bean and in the genus Phaseolus.