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.     

Fast and sensitive screening method for detection of trichothecenes in maize by using protein synthesis inhibition in cultured fibroblasts

A fast and sensitive bioassay with hamster (BHK-21 C13) fibroblasts for the detection of toxic trichothecenes in maize is described. Cells are exposed to pure toxins or crude maize extracts for 30 min. The mixture is then incubated with [1-14C]-leucine for an additional 60 min and the radioactivity incorporated into the protein of the washed cells is determined. The sensitivity of the assay was in the range 1-10 ng/mL (or 50 ppb in maize) for T-2, HT-2, and diacetoxyscirpenol. At least 1000-fold higher concentrations of non-trichothecene mycotoxins and plant toxins were necessary to cause an inhibition of protein synthesis in the cells. Of 24 maize samples tested, 14 gave a positive response in this assay and the presence of trichothecenes could be confirmed chemically in 11 samples. Therefore, the described bioassay is proposed as a useful screening method for cytotoxic trichothecenes in maize.     

Protein-protein interactions during high-moisture extrusion for fibrous meat analogues and comparison of protein solubility methods using different solvent systems

Soy protein, mixed with gluten and starch, was extruded into fibrous meat analogues under high-moisture and high-temperature conditions. The protein solubility of samples collected at different extruder zones and extrudates made with different moistures was determined by 11 extraction solutions consisting of 6 selective reagents and their combinations: phosphate salts, urea, DTT, thiourea, Triton X-100, and CHAPS. Protein solubility by most extractants showed decreasing patterns as the material passed through the extruder, but the solution containing all 6 reagents, known as isoelectric focus (IEF) buffer, solubilized the highest levels and equal amounts of proteins in all samples, indicating that there are no other covalent bonds involved besides disulfide bonds. With regard to relative importance between disulfide bonds and non-covalent interactions, different conclusions could be made from protein solubility patterns, depending on the type of extracting systems and a baseline used for comparison. The observation points out pitfalls and limitation of current protein solubility methodology and explains why controversy exists in the literature. Using the IEF buffer system with omission of one or more selective reagents is considered to be the right methodology to conduct protein solubility study and thus recommended. Results obtained with this system indicate that disulfide bonding plays a more important role than non-covalent bonds in not only holding the rigid structure of extrudates but also forming fibrous texture. The sharpest decrease in protein solubility occurred when the mix passed through the intermediate section of the extruder barrel, indicating formation of new disulfide bonds during the stage of dramatic increase in both temperature and moisture. After this stage, although the physical form of the product might undergo change and fiber formation might occur as it passed through the cooling die, the chemical nature of the product did not change significantly.     

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.     

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.     

Genetic parameters and selection of maize cultivars using Bayesian inference in a multi-trait linear model

Variance components must be obtained to estimate genetic parameters and predict breeding values. In studies which take many traits into account, it is reasonable to use the Bayesian approach for the estimation of genetic parameters. The main goal of the present research was not only to consider the genetic correlations of the examined traits, but above all to estimate unknown genetic parameters and to gain profits from the selection. Bayesian inference was also useful for the selection of the best maize varieties. It was applied to predict genetic values in the multi-traits linear model. Thirteen maize cultivars representing the traits of our interest were studied by means of Bayesian inference. The traits are the number of plants before harvest, the grain yield, the length of the ears, the mass of leaves and the number of ears. The experiment involved a randomised block design with four replications and ten plants per plot. The highest correlation estimates were found between the number of plants before harvest and the number of ears, jointly with the grain yield and the number of ears. Lower correlation estimates were found between the length of the ears and the number of ears as well as the grain yield and the length of the ears. The research confirms that the best varieties to be grown are: Clarica, NK Cooler, Drim and PR 39K13. The Bayesian approach proved to be useful in selection studies, which can further be used to improve the studied genotypes.     

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.     

冬小麦–夏玉米轮作体系不同新型尿素的氮素利用率及去向

  【目的】  以华北平原冬小麦–夏玉米轮作体系为研究对象，探究新型尿素对作物氮素吸收及去向的影响。  【方法】  于2016年10月至2017年9月，在河北省辛集市河北农业大学马庄试验站进行小麦15N田间微区试验，微区面积为1 m2，设置施用普通尿素、普通尿素 + 硝化抑制剂 (Nr)、控失尿素、聚能网尿素和腐植酸尿素5个处理，各施肥处理氮素施用量均为N 225 kg/hm2，并以不施氮肥处理为对照。  【结果】  5个氮肥处理相比，控失尿素处理的冬小麦产量最高，为8123 kg/hm2；腐植酸尿素处理次之，为8083 kg/hm2；再次为聚能网尿素处理，为8049 kg/hm2。控失尿素、腐植酸尿素、聚能网尿素、普通尿素 + Nr、普通尿素的15N当季利用率分别为43.6%、41.1%、37.8%、34.2%、32.2%；控失尿素、腐植酸尿素的15N当季利用率显著高于普通尿素 + Nr和普通尿素处理，聚能网尿素的15N当季利用率显著高于普通尿素，普通尿素 + Nr处理则与普通尿素处理的15当季利用率无显著差异；控失尿素的15N当季利用效果最为突出，较普通尿素15N当季利用率提高了35.4%，腐植酸尿素、聚能网尿素较普通尿素15N当季利用率显著提高了27.6%、17.4%。后茬玉米能吸收利用前茬小麦残留在土壤中的氮素，但后茬玉米的土壤残留15N利用率仅为2.98%～3.62%，4种新型尿素处理间后茬玉米15N利用率无显著性差异。小麦收获后，4种新型尿素均显著提高了土壤上层 (0—40 cm) 硝态氮残留量，有利于后茬玉米对氮素的吸收，减少氮素淋溶的可能性。肥料氮总损失表现为控失尿素、聚能网尿素 < 腐植酸尿素、普通尿素 + Nr < 普通尿素。  【结论】  新型尿素显著促进作物对氮素的吸收利用，减少氮素损失，获得高产。4种新型尿素相比，控失尿素增产增效最为突出，土壤中氮残留少，损失率低，其当季利用率、残留率和损失率分别为43.6%、40.8%和15.6%；腐植酸尿素氮当季利用率仅次于控失尿素，而损失率较高达19.8%；聚能网尿素有利于氮素固持在土壤中，其残留率、损失率分别为46.1%、16.1%；普通尿素 + Nr处理的氮素当季利用率偏低而土壤残留率最高，分别为34.2%和47.4%。     

Accelerated solvent extraction and gas chromatography/mass spectrometry for determination of polycyclic aromatic hydrocarbons in smoked food samples.

Accelerated solvent extraction (ASE) is a new sample extraction method offering a number of advantages such as low pe -extraction cost, reduced solvent and time consumption, and simplified extraction protocols. In this study, the ASE method was applied to the extraction of polycyclic aromatic hydrocarbons (PAHs) from biological samples. For recovery studies, fish tissues and ground pork were used as sample matrices. Sample aliquots fortified with 16 PAHs were extracted by ASE, and the extracts were treated with sulfuric acid and Florisil, followed by gas chromatography-mass spectrometry analysis. The PAH recoveries by the ASE method were found to be comparable with or better than those by Soxhlet extraction. The extraction and quantitation method was then applied to the determination of PAHs in several smoked meat samples obtained from a local market. Up to 12 PAHs were found to be present at concentrations ranging from 3 to 52 ng/g wet sample.     

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.     

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.     

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.