DNA-based identification of spices: DNA isolation, whole genome amplification, and polymerase chain reaction

Usually spices are identified morphologically using simple methods like magnifying glasses or microscopic instruments. On the other hand, molecular biological methods like the polymerase chain reaction (PCR) enable an accurate and specific detection also in complex matrices. Generally, the origins of spices are plants with diverse genetic backgrounds and relationships. The processing methods used for the production of spices are complex and individual. Consequently, the development of a reliable DNA-based method for spice analysis is a challenging intention. However, once established, this method will be easily adapted to less difficult food matrices. In the current study, several alternative methods for the isolation of DNA from spices have been developed and evaluated in detail with regard to (i) its purity (photometric), (ii) yield (fluorimetric methods), and (iii) its amplifiability (PCR). Whole genome amplification methods were used to preamplify isolates to improve the ratio between amplifiable DNA and inhibiting substances. Specific primer sets were designed, and the PCR conditions were optimized to detect 18 spices selectively. Assays of self-made spice mixtures were performed to proof the applicability of the developed methods.     

BSE infectivity in jejunum, ileum and ileocaecal junction of incubating cattle

ABSTRACT: To establish bovine spongiform encephalopathy (BSE) public health protection measures it is important to precisely define the cattle tissues considered as specified risk materials (SRM). To date, in pre-clinical BSE infected cattle, no evidence of the BSE agent had been found in the gut outside of the ileal Peyer's Patches. This study was undertaken to determine when and where the pathological prion protein (PrPSc) and/or BSE infectivity can be found in the small intestine of cattle 4 to 6 months of age, orally challenged with BSE. Samples of the jejunum, the ileum and the ileocaecal junction from 46 BSE infected cattle, culled from 1 up to 44 months post infection (mpi) were examined by immunohistochemistry. Samples from cattle 8 mpi to 20 mpi were additionally studied by PTA Western blot, rapid tests, and by mouse (TgbovXV) bioassay. In doing so nearly all of the cattle, from 4 up to 44 mpi, had detectable amounts of PrPSc and/or infectivity in the distal ileum. In the distal ileum clear time-dependent variations were visible concerning the amount of PrPSc, the tissue structures affected, and the cells involved. BSE infectivity was found not only in the ileum and ileocaecal junction but also in the jejunum. The systematic approach of this study provides new data for qualitative and quantitative risk assessments and allows defining bovine SRM more precisely.     

Simultaneous determination and differentiation of glycidyl esters and 3-monochloropropane-1,2-diol (MCPD) esters in different foodstuffs by GC-MS

The aim of this study was the development of a method for the simultaneous determination and differentiation of fatty acid esters of 3-monochloropropane-1,2-diol (3-MCPD esters) and glycidol (glycidyl esters) in different foodstuffs. The esters were isolated from fat-rich food samples using a single extraction step and separated from interfering substances. For differentiation of 3-MCPD esters and glycidyl esters the glycidol moiety was converted into 3-methoxypropane-1,2-diol (3-MPD) by acidic alcoholysis. Subsequent determination was achieved by isotope dilution GC-MS after transesterification using an isotope-labeled 3-MCPD ester as internal standard. During optimization of the procedure, critical parameters affecting simultaneous determination and differentiation of these analytes were investigated. Rapid ester cleavage and derivatization at ambient temperature proved to be essential for the simultaneous determination of these analytes. The method was validated for various fat-rich foodstuffs such as bakery products, sweets, gravy, and soup powders as well as edible fats and oils. LODs of 8 and 15 μg/kg (fat-rich foodstuffs) as well as 50 and 65 μg/kg (edible oils and fats) were obtained for 3-MCPD esters and glycidyl esters, respectively. Recoveries for 3-MCPD esters and glycidyl esters ranged within 98 ± 4 and 88 ± 2% in all tested foodstuffs (0.05-2.5 mg/kg) and within 99 ± 16 and 93 ± 13% for edible oils and fats (0.15-3 mg/kg) over a wide concentration range. These results proved an accurate and differentiated determination of 3-MCPD esters and glycidyl esters with successful application to the fast screening of samples, avoiding tedious and laborious sample preparation.     

Serosurvey for selected viral agents in white rhinoceros (Ceratotherium simum) in Kruger National Park, 2007

One hundred serum samples collected from free-ranging white rhinoceros (Ceratotherium simum) in Kruger National Park (KNP) during the 2007 capture season were selected for measurement of antibody levels to several different vector-borne viral agents. These infectious diseases were chosen to compare with an earlier serosurvey that had been conducted in KNP in rhinos during 1987-1997. Positive antibody titers were found against epizootic hemorrhagic disease (EHD) of deer (8%), Bluetongue (BT) (1%), and Rift Valley fever (RVF) (49%). However, none of the 100 animals tested had detected antibody levels to African horse sickness (AHS). These values were in sharp contrast to those measured in the 1987-1997 survey in KNP white rhinos (AHS 60%, EHD 30%, BT 37%, RVF 0%). Vector-borne viral infection prevalence in white rhinos in the same geographical location appears to vary over time and may be important for monitoring presence of pathogens in an ecosystem.     

Automatic adjustment of harrowing intensity in cereals using digital image analysis

Precision farming technologies were implemented into a commercial harrow to increase selectivity of weed harrowing in spring cereals. Digital cameras were mounted before and after the harrow measuring crop cover. Crop soil cover (CSC) was computed out of these two images. Eight field experiments were carried out in spring cereals. Mode of harrowing intensity was changed in four experiments by speed, number of passes and tine angle. Each mode was varied in five intensities. In four experiments, only intensity of harrowing was changed. Weed control efficacy (WCE) and CSC were measured immediately after harrowing. Crop recovery was assessed 14 days after harrowing. Modes of intensity were not significantly different. However, intensity had significant effects on WCE and CSC. Cereals recovered from 10% CSC, and selectivity was in the constant range at 10% CSC. Therefore, 10% CSC was the threshold for the decision algorithm. If the actual CSC was below 10% CSC, intensity was increased. If the actual CSC was higher than 10%, intensity was decreased. Image analysis, decision support system and automatic control of harrowing intensity by hydraulic adjustment of tine angle were installed on a controller mounted on the harrow. The new system was tested in an additional field study. Threshold values for CSC were set at 10%, 30% and 60%. Automatic tine angle adjustment precisely realised the three different CSC values with variations of 1.5% to 3%. This development contributes to selective weed control and supports farmers during harrowing.     

Utility of Metabolomics toward Assessing the Metabolic Basis of Quality Traits in Apple Fruit with an Emphasis on Antioxidants

A gas chromatography-mass spectrometry approach was employed to evaluate the use of metabolite patterns to differentiate fruit from six commercially grown apple cultivars harvested in 2008. Principal component analysis (PCA) of apple fruit peel and flesh data indicated that individual cultivar replicates clustered together and were separated from all other cultivar samples. An independent metabolomics investigation with fruit harvested in 2003 confirmed the separate clustering of fruit from different cultivars. Further evidence for cultivar separation was obtained using a hierarchical clustering analysis. An evaluation of PCA component loadings revealed specific metabolite classes that contributed the most to each principal component, whereas a correlation analysis demonstrated that specific metabolites correlate directly with quality traits such as antioxidant activity, total phenolics, and total anthocyanins, which are important parameters in the selection of breeding germplasm. These data sets lay the foundation for elucidating the metabolic basis of commercially important fruit quality traits.     

Aggregate stability and physical protection of soil organic carbon in semi‐arid steppe soils

Spatial inaccessibility of soil organic carbon (SOC) for microbial decay within soil aggregates is an important stabilization mechanism. However, little is known about the stability of aggregates in semiarid grasslands and their sensitivity to intensive grazing. In this study, a combined approach using soil chemical and physical analytical methods was applied to investigate the effect of grazing and grazing exclusion on the amount and stability of soil aggregates and the associated physical protection of SOC. Topsoils from continuously grazed (CG) and ungrazed sites where grazing was excluded from 1979 onwards (UG79) were sampled for two steppe types in Inner Mongolia, northern China. All samples were analysed for basic soil properties and separated into free and aggregate‐occluded light fractions (fLF, oLF) and mineral‐associated fractions. Tensile strength of soil aggregates was measured by crushing tests. Undisturbed as well as artificially compacted samples, where aggregates were destroyed mechanically by compression, were incubated and the mineralization of SOC was measured. For undisturbed samples, the cumulative release of CO₂‐C was greater for CG compared with UG79 for both steppe types. A considerably greater amount of oLF was found in UG79 than in CG soils, but the stabilities of 10–20‐mm aggregates were less for ungrazed sites. Compacted samples showed only a slightly larger carbon release with CG but a considerably enhanced mineralization with UG79. We assume that the continuous trampling of grazing animals together with a smaller input of organic matter leads to the formation of mechanically compacted stable ‘clods’, which do not provide an effective physical protection for SOC in the grazed plots. In UG79 sites, a greater input of organic matter acting as binding agents in combination with an exclusion of animal trampling enhances the formation of soil aggregates. Thus, grazing exclusion promotes the physical protection of SOC by increasing soil aggregation and is hence a management option to enhance the C sequestration potential of degraded steppe soils.     

Physical properties of a Luvisol for different long‐term fertilization treatments: II. Microscale behavior and its relation to the mesoscale

We determined the impact of different fertilization, namely organic vs. mineral fertilization, on the mesoscale parameter cyclic compressibility as well as on rheology of soil samples as a microscale parameter and how these parameters are related. Therefore, undisturbed samples were taken from a long‐term fertilization trial at the Dikop farm near Bonn (Germany) and tested for their mechanical and hydraulic properties. This paper examines the sensitivity of the soil towards cyclic loading (mesoscale) and oscillatory shearing at the microscale by means of an amplitude sweep test and the resulting parameter maximum shear stress. Fertilization increased cyclic compressibility and thus revealed structural weakness of fertilized soil samples, so did shear stress at the microscale. The main reason for this was a decrease in bulk density in the wake of fertilization. However, within the range of fertilized soil samples, the soil structure became less susceptible towards cyclic loading and oscillatory shearing, respectively, the more organic matter the soil contained (equivalent to the fertilization level). This was assumedly caused by enhanced cementation due to organic substances that could partly substitute the direct grain–grain contacts generally contributing to soil strength. The similar behavior of cyclic compressibility and maximum shear stress enabled a first approach to relate soil mechanical parameters at the microscale to those at the mesoscale.     

Application of ultrasound to disperse soil aggregates of high mechanical stability

Questions remain about the exact ultrasonic energy level that is required to effectively disperse soil aggregates and to what extent this is accompanied by physical damage to individual soil particles. We found maximum aggregate dispersion at energy levels of 1500 J cm^–3 and no evidence for the disintegration of particles < 20 μm even at that energy level. Our findings suggest that sonication at energies much greater than those applied conventionally can disperse aggregates of high mechanical stability.     

Suitability of inorganic and organic amendments for in situ immobilization of Cd,Cu, and Zn in a strongly contaminated Kastanozem of the Mashavera valley,SE Georgia. I. Effect of amendments on metal mobility and microbial activity in soil

The aim was to find an adequate, cost‐efficient in situ remediation technique for the Mashavera valley, a mining area in SE Georgia heavily contaminated with Cd, Cu, and Zn. A 12‐month experiment was conducted to test: iron grit, natural zeolite, biochar, and Divergan® (a scavenger) for soil melioration. The amendments were added in different concentrations to the topsoil of a Kastanozem. Mobile metal concentrations decreased with increasing concentrations of amendments in the sequence Divergan® >> iron grit ≈ natural zeolite > biochar. In the same order amendments enhanced activities of soil microbial respiration, alkaline phosphatase, and dehydrogenase; microbial C also followed this trend. A sequential extraction confirmed a shift from easily mobilized to heavily bound fractions. The addition of 2% (w/w) of Divergan® was sufficient to lower mobile trace metal concentrations below German thresholds by chemisorption, and soil microbial activity was significantly increased. The effects of all other treatments were at a much lower level and not found suitable due to needed application rates.