Extraktion eines kupferbelasteten Bodens unter Verwendung eines aminosäurehaltigen Reststoffhydrolysats. 2. Elutionsdynamik und Bilanzierung der Aminosäuren nach kontinuierlichem Eintrag in Bodensäulen

Extraction of a copper contaminated soil material by the percolation of an amino acid containing residue hydrolysate. 2. Time course of amino acid elution and input/output balance of amino acids During 16 days an amino acid containing blood meal hydrolysate (amino acid concentration: 188 mMol·L^?1) was percolated through a column packed soil material (soil content per column: 4.1 kg dry weight, four parallels). The copper contaminated material (soil type: Typic Udifluvent, soil texture: sandy loam, loamy sand) was sampled from an area formerly used for cultivation of hop (Humulus lupus). Besides the investigation of the copper liberation the experiments aimed to determine the elution dynamic and input/output balance of amino acids (time span for amino acids balance 14 days). In total 11.7 L of hydrolysate, containing 2.2 Mol of amino acids, were introduced into each column. The mean amino acid output with the column effluent was 1.13 Mol. This corresponds to an elution degree of 51.2%, related to the sum of applicated amino acids, and to a mean substance specific elution degree of 48.4% reflecting the elution of 15 compounds. The substance specific elution ranged from 9.6% (serine) to 75.5% (valine). The highest concentrations of serine and threonine were determined in the effluents after two days, whereas the histidine concentration was highest at the last sampling. The differences in the percolation properties of the amino acids are discussed in terms of important retention and elimination processes (biodegradation, ad-/desorption, intercalation).     

Untersuchung zur Anwendbarkeit von Sorptionskoeffizienten für die Simulation der Verlagerung von 2, 4, 5-T und LAS in Böden

Investigations for the use of sorption coefficients for simulation of the transport of 2, 4, 5-T and LAS in soils The use of sorption coefficients for the prediction of the transport of 2, 4, 5-trichlorophenoxy-acetic acid (2, 4, 5-T) and of n-dodecylbenzene-sulfonate (LAS) was investigated for four different soil types with the simulation model EXSOL. Linear equilibrium sorption coefficients of 2, 4, 5-T determined in batch studies are not suited for the prediction of the transport wheras values derived from the retention in soil columns better fit the experimental profiles of 2, 4, 5-T in the field. Sorption coefficients for LAS from batch studies with a soil-solution ration of 1:2 and a shaking period of 4 h gave the best results for prediction of LAS transport in the field.     

Regenwürmer als Bioindikatoren für Schwermetallbelastungen von Böden unter Freilandbedingungen

The use of earthworms in monitoring soil pollution by heavy metals Total heavy metal contents (HNO₃-soluble) and exchangeable fractions (Ca(NO₃)₂-soluble) of Pb, Zn and Cd were measured in soils, litter layers and earthworms (dry masses) from forest, arable and pasture sites in a transect of the main wind direction and varying distances (1.5, 5.4, 11.4, 15.6 km) to a lead smelter near Bad Ems, Germany. Additionally, cast materials of Lumbricus terrestris-individuals were collected from the surface of the pasture sites. In the observed area total soil contents of Pb and Cd exceeded the C-level and total contents of Zn the B-level of the “Netherland-list”. Heavy metal contents in soils and earthworms decreased with increasing distance to the smelter. Pb showed the best correlation. Correlation between total contents of Pb and Cd in soils and earthworms were significant (rs = 0.66; p < 0.05 and rs = 0.67; p < 0.01, respectively). The uptake of heavy metals by Lumbricus rubellus, L. terrestris and Aporrectodea caliginosa was metal specific rather than species specific with factors of accumulation being <1 (Pb), 2.7–7.6 (Zn) and 19.5–85.5 (Cd). The heavy metal contents of the observed cast materials signified the different transfer of elements from soil material via earthworm individuals to the faeces. In the cast materials the amounts of Pb were high and the amounts of Zn and Cd were low. This indicates a high accumulation rate for Zn and especially for Cd in the tissues of the observed earthworm individuals. The present data support the necessity of ecotoxicological threshold levels.     

Bestimmung von Mikronährstoffen und Schwermetallen in Böden mit dem Verfahren der Elektro‐Ultrafiltration (EUF) durch Zugabe von DTPA

With the electro–ultrafiltration (EUF) technique, the plant availability of several plant nutrients in soils can be characterized. The basic principle of EUF is that an electric field is induced using platinum electrodes. Ions in the soil suspension move either to the cathode or to the anode and are filtrated through ultra‐membrane filters. In the standard EUF procedure, two extractions steps are used: 30 min at 20°C and 5 min at 80°C. However, the determination of micronutrients and heavy metals with the standard EUF procedure is not possible, because the solubility of these elements in water is low and most of the watersoluble elements are precipitated when passing the platinum electrodes. The addition of DTPA, a well known complexing agent, during a third EUF fraction (5 min at 80°C) enables extraction of micronutrients and heavy metals. Highest concentrations in the 33 soils of the study were found for iron, followed by zinc, manganese, lead, copper, and nickel. Lower concentrations were obtained for cobalt, chromium, cadmium, and molybdenum. For two soils, the EUF/DTPA procedure was compared to CaCl₂/DTPA and EDTA soil extraction methods, showing that higher or comparable amounts were found with CaCl₂/DTPA and much higher amounts with the EDTA method. These results reveal that the EUF/DTPA technique in principle can be used for the determination of plant‐available micronutrients and heavy metals. However, in a next step the relationship between EUF/DTPA‐extractable elements and their availability for plants needs to be quantified.     

Constituents in evening primrose oil with radical scavenging,cyclooxygenase, and neutrophil elastase inhibitory activities

Cold-pressed, non-raffinated evening primrose oil was found to contain lipophilic radical scavengers. A highly enriched fraction of these compounds could be obtained from the oil by extraction with aqueous ethanol and subsequent liquid-liquid partitioning with petroleum. LC-DAD-MS analysis revealed that the fraction contained three aromatic compounds with identical UV and ESI-MS spectra. The compounds were isolated by RP-HPLC and their structures established by chemical and spectroscopic means as 3-O-trans-caffeoyl derivatives of betulinic, morolic, and oleanolic acid. The morolic acid derivative was a new compound. The three esters exhibited pronounced radical scavenging activity against the stable 2,2-diphenyl-1-picrylhydrazyl radical and were potent inhibitors of neutrophil elastase and cyclooxygenase-1 and -2 in vitro. Commercial samples of evening primrose oils contained only traces of these lipophilic antioxidants.     

Differentiation between lutein monoester regioisomers and detection of lutein diesters from marigold flowers (Tagetes erecta L.) and several fruits by liquid chromatography-mass spectrometry.

Liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCIMS) was employed for the identification of eight lutein monoesters, formed by incomplete enzymatic saponification of lutein diesters of marigold (Tagetes erecta L.) by Candida rugosa lipase. Additionally, the main lutein diesters naturally occurring in marigold oleoresin were chromatographically separated and identified. The LC-MS method allows for characterization of lutein diesters occurring as minor components in several fruits; this was demonstrated by analysis of extracts of cape gooseberry (Physalis peruviana L.), kiwano (Cucumis metuliferus E. Mey. ex Naud.), and pumpkin (Cucurbita pepo L.). The assignment of the regioisomers of lutein monoesters is based on the characteristic fragmentation pattern: the most intense daughter ion generally results from the loss of the substituent (fatty acid or hydroxyl group) bound to the epsilon-ionone ring, yielding an allylic cation. The limit of detection was estimated at 0.5 microg/mL with lutein dimyristate as reference compound. This method provides a useful tool to obtain further insight into the biochemical reactions leading to lutein ester formation in plants.     

Minimally-invasive catheterization of the portal, hepatic and cranial mesenteric veins and the abdominal aorta for quantitative determination of hepatic metabolism in dairy cows

The objective of this study was to establish a minimally-invasive, ultrasound (US)-guided technique for the placement of indwelling catheters into the portal, hepatic, and cranial mesenteric veins as well as the abdominal aorta. Catheters were placed in eight healthy dairy cows on day 1. The patency of catheters was tested daily until day 14 when a necropsy was carried out. On day 6, energy intake and hepatic net output of glucose, removal of lactate, and oxygen were determined in seven cows. Post mortem examination revealed that all implanted catheters were in the intended locations. Loss of patency in one portal vein catheter on day 9 was attributable to a fibrin clot. Significant correlations were found between mean energy intake and mean hepatic plasma flow (r=0.91; P=0.004), hepatic glucose output (r=0.81; P=0.027) and hepatic removal of lactate (r=-0.70; P=0.08) and oxygen (r=-0.77; P=0.039), as well as between hepatic glucose net output and removal of lactate (r=-0.92; P=0.004). Minimally-invasive, US-guided transcutaneous catheter placement into the cranial mesenteric, portal and hepatic veins as well as the technique for catheterization of the abdominal aorta appear to be safe, and suitable for studies of quantitative hepatic metabolism in cattle.     

Use of isotope-labeled aflatoxins for LC-MS/MS stable isotope dilution analysis of foods

Aflatoxins are a group of very carcinogenic mycotoxins that can be found on a wide range of food commodities including nuts, cereals, and spices. In this study, the first LC-MS/MS stable isotope dilution assay (SIDA) for the determination of aflatoxins in foods was developed. The development of this method was enabled by easily accessible isotope-labeled (deuterated) aflatoxins B2 and G2, which were synthesized by catalytic deuteration of aflatoxin B1 and G1, purified, and well-characterized by NMR and MS. All four aflatoxins of interest (B1, B2, G1, and G2) were quantified in food samples by using these two labeled internal standards. The response factors (RF) of the linear calibrations were revealed to be matrix independent for labeled aflatoxin B2/aflatoxin B2 and labeled aflatoxin G2/aflatoxin G2. For labeled aflatoxin B 2/aflatoxin B 1 and labeled aflatoxin B2/aflatoxin G1 matrix-matched calibration was performed for the model matrices almonds and wheat flour, showing significant differences of the RFs. Limits of detection (LOD) were determined by applying a statistical approach in the presence of the two model matrices, yielding 0.31 microg/kg (aflatoxin B1), 0.09 microg/kg (aflatoxin B2), 0.38 microg/kg (aflatoxin G1), and 0.32 microg/kg (aflatoxin G2) for almonds (similar LODs were obtained for wheat flour). Recovery rates were between 90 and 105% for all analytes. Coefficients of variation (CV) of 12% (aflatoxin B1), 3.6% (aflatoxin B2), 14% (aflatoxin G1), and 4.8% (aflatoxin G2) were obtained from interassay studies. For further validation, a NIST standard reference food sample was analyzed for aflatoxins B1 and B2. The method was successfully applied to determine trace levels of aflatoxins in diverse food matrices such as peanuts, nuts, grains, and spices. Aflatoxin contents in these samples ranged from about 0.5 to 6 microg/kg.     

High-level expression of recombinant beta-galactosidases in Lactobacillus plantarum and Lactobacillus sakei using a Sakacin P-based expression system

This work presents the cloning and expression of the genes encoding heterodimeric beta-galactosidases from Lactobacillus reuteri L103, Lactobacillus acidophilus R22, Lactobacillus plantarum WCFS1, and Lactobacillus sakei Lb790. These enzymes consist of two subunits of approximately 73 and 35 kDa, which are encoded by two overlapping genes, lacL and lacM, respectively. We have cloned these genes into the lactobacillal expression vectors pSIP403 and pSIP409, which are based on the sakacin P operon of L. sakei ( S?rvig et al. Microbiology 2005, 151, 2439- 2449 ), and expressed them in the host strains L. plantarum WCFS1 and L. sakei Lb790. Results varied considerably, ranging from 2.23 to 61.1 U/mg of beta-galactosidase activity, depending on the origin of the lacLM genes, the host strain, and the expression vector used. Highest expression levels were obtained in a laboratory cultivation of L. plantarum WCFS1 harboring the plasmid pEH3R containing the lacLM gene from L. reuteri L103. These cultivations yielded approximately 23 000 U of beta-galactosidase activity per liter, corresponding to the formation of roughly 100 mg of recombinant protein per liter of fermentation medium, and beta-galactosidase levels amounted to 55% of the total intracellular protein of the host organism. To further verify the suitability of this expression system, recombinant beta-galactosidase from L. reuteri was purified to apparent homogeneity. The properties of the purified enzyme were essentially identical with the properties of purified native beta-galactosidase from L. reuteri L103. The presented results lead the way to efficient overproduction of beta-galactosidase in a food-grade expression system, which is of high interest for applications in food industry.     

Upscaling spatio‐temporal patterns of cation fluxes in acid forest soils

One main problem with current research on spatio‐temporal modeling of ion fluxes in forest soils is the separation of space and time effects in the soil‐monitoring concept. This article describes an approach to overcome this weakness. Time trends of point information on soil‐solution data (base‐cation concentrations and fluxes) are scaled by linking them to soil‐chemical data which is available in higher spatial resolution and can be upscaled to an area base. This approach is based on a combined evaluation of bulk soil and soil‐solution data using both statistical and process‐oriented methods. Multiple‐linear‐regression analyses coupled with geostatistics were developed to predict spatial patterns of exchangeable cation percentages. In a second step, empirical ion‐distribution coefficients were adapted according to Gapon using data of suction‐cup plots and bulk‐soil samples. Seasonally adjusted time‐series data of soil‐solution chemistry were then connected with the maps of the predicted exchangeable‐cation percentages by means of the Gapon equations. This evaluation step provided both time‐ and space‐dependent maps of cation concentrations in the soil solution. Finally, using the results of a water‐budget model it was possible to derive spatio‐temporal patterns of soil cation fluxes. Methodological limitations and the results of verification processes are discussed. The methods described can only be used in acidic soils and should not be used in soil layers rich in humus, since adsorption to C compounds differs from adsorption to clay minerals. The time increments of the models should be not shorter than yearly in order to suppress annual periodicity. Although the Gapon equations were not based on laboratory‐determined exchange solutions at quasi‐equilibrium, but rather on field data from the suction‐cup technique, the exchangeable‐cation percentages showed steady functions of selectivity coefficients. The methods tested at a watershed scale may be flexible enough to be applied at other scales as well.