Development and transferability of two multiplexes nSSR in Scots pine (Pinus sylvestris L.)

Single sequence repeat(SSR) multiplexing is a semi high-throughput PCR methodology for the analysis of multiple SSRs.We developed two SSR multiplexes selected from SSR loci previously reported in the pine literature and tested the transferability of both SSR multiplexes in nine other pine species.We tested 234 nuclear SSR loci(n SSRs) previously described in the pine literature and selected ten n SSRs following the simple criteria of interpretability and reproducibility.Selected nuclear loci were divided into two n SSRs multiplex sets and their amplification was optimized for three different multiplex PCR methods based on:(a) a custom PCR protocol,(b) a custom protocol with hotstart taq polymerase,and(c) a commercially available kit for SSR multiplexing.To validate their performance,the level of genetic diversity was assessed in three Scots pine natural populations(Hungary,northern Sweden and southern Sweden).In addition,we also tested the transferability of these multiplexes in nine other pine species.We have developed two n SSRs multiplexes of five loci each that will contribute to reduce the costs of n SSR scoring,while increasing the capacity of n SSR loci analysis.Amplification was successful in all three populations(94 % success) and the level of polymorphism(7.1 alleles/marker) was similar to that previously reported for other Scots pine natural populations.Transferability of both multiplexes was successful for those pine species closely related to Scots pine.     

Soil and Water Contamination Levels in an Out-of-Service Oil Distribution and Storage Station in Michoacan,Mexico

An oil distribution and storage station (ODSS) in Michoacan, Mexico was active from 1963 to 1999. Out-of-service since then, it is now subject to a dismantling process. From the results of this study it was concluded that ODSS soil is contaminated mainly with TPH, BTEX, MTBE, Pb, Fe, Cr, and Zn. Total petroleum hydrocarbons and metal contamination is clearly localised in three main area: in front of the TV-284 storage tank, along the area where the train railway was once situated, and the zone near the pumping house. Pb was detected in groundwater in concentrations higher than 0.025 mg l^-1. A health risk assessment (HRA) was carried out. From the HRA results, it was proposed to reduce the benzene, iron, MTBE, lead, and xylene concentrations from their current values to 0.0072, 6,000, 0.21, 400, and 100 mg kg^-1, respectively. Total petroleum hydrocarbon concentration must be reduced from 59,000 to 2,000 mg kg^-1. Remediation of the ODSS soil with regard to the high TPH, MTBE, Pb, Cr and benzene concentrations appears feasible and will allow the use of the old ODSS with new soil uses at middle and long term.     

Remediation of contaminated soil using soil washing and biopile methodologies at a field level

Background, Aims and Scope  An out-of-service oil distribution and storage station (ODSS), which operated from 1966 to 2000 in Mexico, is contaminated mainly by gasoline and diesel, showing the presence of methyl-tert-butyl-ether, benzene, toluene, ethyl benzene, and xylenes. Nine of the 16 polycyclic aromatic hydrocarbons were found, as well as Fe, Pb, V, and Zn. The health risk assessment suggested the necessity of reducing of three PAHs [benzo(a)anthracene, benzo(a)pyrene, and benzo-(b)fluoranthene], and vanadium. The aim of this work is to show that soil washing (on-site) and biopiles are excellent remediation methodologies to treat soils contaminated with petroleum derivates and metals. Applying them, it is possible to reach the goal value of 2,000 mg TPH/kg in a few months, as requested by Mexican legislation. Methods  More than 140 m³ were excavated from the ODSS. Three soil-washing dishes were built. 1540 m³ were treated by soil washing using a nonionic surfactant. A 100 m³ biopile was built to study the system capabilities in the biodegradation of around 4,500 mg/kg of TPH using the autochthonous microflora. Results and Discussion  The soil washing, average TPH-removal value was 83%, but values up to ca. 93% were observed. Removal values resulted in a function of the TPH initial values. Biopile (100 m³) worked during 66 days, reaching a TPH-removal value of 85%. At the end of the processes, no PAHs were detected. The contaminated soil was treated successfully, reaching the legislation limits (TPH values under 2,000 mg/kg, and a significant reduction in PAH concentrations). Conclusion and Recommendation  Both systems are suitable for remediation purposes, achieving high removal efficiencies at short and medium stages. It is highly recommended to proceed with soil washing studies, identifying new products, and mixtures, which could reduce costs and assure optimum operation.     

Influence of the addition of rosemary essential oil on the volatiles pattern of porcine frankfurters

The effect of the addition of increasing levels of rosemary essential oil (150, 300, and 600 mg/kg) on the generation of volatile compounds in frankfurters from Iberian and white pigs was analyzed using solid-phase microextraction coupled to gas chromatography and mass spectrometry (SPME-GC-MS). Lipid-derived volatiles such as aldehydes (hexanal, octanal, nonanal) and alcohols (pentan-1-ol, hexan-1-ol, oct-1-en-3-ol) were the most abundant compounds in the headspace (HS) of porcine frankfurters. Frankfurters from different pig breeds presented different volatile profiles due to their different oxidation susceptibilities as a likely result of their fatty acid composition and vitamin E content. Rosemary essential oil showed a different effect on the generation of volatiles depending on the type of frankfurter in which they were added. In frankfurters from Iberian pigs, the antioxidant effect of the essential oil improved with increasing levels, showing the highest activity at 600 mg/kg. In contrast, 150 mg/kg of the essential oil improved the oxidative stability of frankfurters from white pigs, whereas higher levels led to no effect or a prooxidant effect. The activity of the essential oil could have been affected by the different fatty acid compositions and vitamin E contents between types of frankfurters. SPME successfully allowed the isolation and analysis of volatile terpenes from frankfurters with added rosemary essential oil including alpha-pinene, beta-myrcene, l-limonene, (E)-caryophyllene, linalool, camphor, and 1,8-cineole, which might contribute to the aroma characteristics of frankfurters.     

Fermentation of sugar beet waste by Aspergillus niger facilitates growth and P uptake of external mycelium of mixed populations of arbuscular mycorrhizal fungi

Sugar beet waste has potential value as a soil amendment and this work studied whether fermentation of the waste by Aspergillus niger would influence the growth and P uptake of arbuscular mycorrhizal (AM) fungi. Plants were grown in compartmentalised growth units, each with a root compartment (RC) and two lateral root-free compartments (RFC). One RFC contained untreated soil while the other RFC contained soil, which was uniformly mixed with sugar beet waste, either untreated (SB) or degraded by A. niger (ASB) in a rock phosphate (RP)-supplied medium. The soil in each pair of RFC was labelled with ³³P and ³²P in order to measure P uptake by the AM fungal mycelium, of which length density was also measured. Whole cell fatty acid (WCFA) signatures were used as biomarkers of the AM fungal mycelium and other soil microorganisms. The amount of biomarkers of saprotrophic fungi and both Gram-positive and Gram-negative bacteria was higher in SB than in ASB treatments. Whilst ASB increased growth and activity of AM mycelium, SB had the opposite effect. Moreover, shoot P content was increased by the addition of ASB, and by inoculation with AM fungi. Modification of soil microbial structure and production of exudates by A. niger, as a consequence of fermentation process of sugar beet waste, could possibly explain the increase of AM growth in ASB treatments. On the other hand, the highest P uptake was a result of the solubilisation of rock phosphate by A. niger during the fermentation.     

Efficient one pot extraction and depolymerization of grape (Vitis vinifera) pomace procyanidins for the preparation of antioxidant thio-conjugates

Antioxidant thio-conjugates were obtained from white grape pomace by a clean and efficient one pot extraction and depolymerization method using water and cysteamine hydrochloride. To evaluate the potential of grape pomaces of different origins as sources of proanthocyanidins and conjugates, we conducted varietal comparative studies of polyphenol content, antioxidant power, and procyanidin composition. Xarel.lo proved to be the richest source of polyphenols. The total conversion into the conjugates was as high as 8 g/kg of Xarel.lo grape pomace, with a 50-fold excess of cysteamine, and 3 g/kg, with a 5-fold excess of cysteamine. After purification by preparative cation exchange and reversed phase high-performance liquid chromatography, 17 g of 63% pure 4beta-(2-aminoethylthio)epicatechin (acetate salt) was obtained from 35 kg of moist pomace. The procedure described here will make the antioxidant thio-derivatives efficiently available directly from raw plant byproducts such as grape pomace.     

PCR-ELISA for the semiquantitative detection of Nile perch (Lates niloticus) in sterilized fish muscle mixtures

A PCR-ELISA technique was developed for the semiquantitative detection of Nile perch (Lates niloticus) in experimentally sterilized fish muscle mixtures. Specific oligonucleotides derived from the 5S rDNA gene of Nile perch were selected. A forward primer, together with a reverse digoxigenin-labeled primer, permitted the amplification of specific 185 bp DNA fragments showing DNA intensities proportional to the contents of Nile perch muscle tissue in the fish mixtures. A biotinylated probe immobilized onto streptavidin-coated microplates was used to capture the digoxigenin-labeled fragments that were detected with peroxidase antidigoxigenin conjugate. Subsequent enzymatic conversion of substrate gave distinct absorbance differences when assaying fish binary mixtures containing different percentages of Nile perch muscle.     

Fate of fumonisin B(1) in corn kernel steeping water containing SO(2)

Six 100 ppm fumonisin B(1) (FB(1)) solutions were prepared by dissolving pure standard in six different solvents containing SO(2). Two of the solvents contained 0.2 or 0.4% SO(2) in distilled water. The other four solvents were obtained by steeping corn kernels at 60 degrees C in a 0.2% SO(2) aqueous solution for 6, 12, 24, or 48 h. After the addition of FB(1), all solutions were maintained at 60 degrees C for 7 days. Fumonisin B(1) content in each solution was determined in triplicate by HPLC. Steeping corn kernels in 0.2% solution at 60 degrees C for 6 h seems to be the most effective treatment to decrease the amount of FB(1).     

Determination of ascorbic acid and carotenoids in food commodities by liquid chromatography with mass spectrometry detection

Two methods, one to determine ascorbic acid and one to determine lycopene and beta-carotene, in vegetables and fruits by liquid chromatography coupled with mass spectrometry (LC-MS) have been established. The chromatographic separation of the studied compounds and their MS parameters were optimized to improve selectivity and sensitivity. In both methods, separation was carried out with two coupled columns, first a C(18) and then a dC(18), using as mobile phase 70% methanol (0.005% acetic acid) and 30% acetic acid 0.05% for ascorbic acid determination and a mixture of methanol, tetrahydrofuran, and acetonitrile (60:30:10 v/v/v) for carotenoid analysis in isocratic mode. The molecular ion was selected for the quantification in selective ion monitoring (SIM) mode. Ascorbic acid was detected with electrospray ionization probe (ESI) in negative mode, while chemical ionization atmospheric pressure (APCI) in positive mode was used for the target carotenoids. The methodology for ascorbic acid analysis is based on an extraction with polytron using methanol and a mixture of methaphosphoric acid and acetic acid. Extraction of the carotenoids was carried out with tetrahydrofuran/methanol (1:1) (v/v). The proposed methods were applied, after their corresponding validations, to the analysis of four varieties of tomatoes, tomato in tin enriched and dried tomato, and to the analysis of mango and kiwi fruits, to compare the content in these compounds. Moreover, the influence of the process of freezing and the effect that the manipulation/preservation has in the content of ascorbic acid in tomato have also been studied.     

Strecker-type degradation produced by the lipid oxidation products 4,5-epoxy-2-alkenals

Strecker degradation is one of the most important reactions leading to final aroma compounds in the Maillard reaction. In an attempt to clarify whether lipid oxidation products may be contributing to the Strecker degradation of amino acids, this study analyzes the reaction of 4,5-epoxy-2-alkenals with phenylalanine. In addition to N-substituted 2-(1-hydroxyalkyl)pyrroles and N-substituted pyrroles, which are major products of the reaction, the formation of both the Strecker aldehyde phenylacetaldehyde and 2-alkylpyridines was also observed. The aldehyde, which was produced at 37 degrees C-as could be determined by forming its corresponding thiazolidine with cysteamine-and pH 6-7, was not produced when the amino acid was esterified. This aldehyde is suggested to be produced through imine formation, which is then decarboxylated and hydrolyzed. This reaction also produces a hydroxyl amino derivative, which is the origin of the 2-alkylpyridines identified. All these data indicate that Strecker-type degradation of amino acids is produced at 37 degrees C by some lipid oxidation products. This is a new proof of the interrelations between lipid oxidation and Maillard reaction, which are able to produce common products by analogue mechanisms.