Determination of tetracycline antibiotic residues in edible swine tissues by liquid chromatography with spectrofluorometric detection and confirmation by mass spectrometry

A sensitive and specific method is described for the simultaneous determination of oxytetracycline, tetracycline (TC), and chlortetracycline residues in edible swine tissues, by combining liquid chromatography with spectrofluorometric and mass spectrometry detection. The procedure involved a preliminary extraction with EDTA-McIlvaine buffer acidified at pH 4.0, followed by solid-phase extraction cleanup using a polymeric sorbent. The liquid chromatography analysis was performed with spectrofluorometric detection after postcolumn derivatization with magnesium ions. The limits of quantification were 50 microg/kg for muscle and 100 microg/kg for kidney tissues. The recovery values were greater than 77.8% for muscle and 65.1% for kidney. The method has been successfully used for the quantification of tetracyclines in swine tissues samples. The selective liquid chromatography mass spectrometric analysis for confirmation of oxytetracycline in one positive swine muscle sample was made by atmospheric pressure chemical ionization (APCI). The APCI mass spectra of the TCs gave the protonated molecular ion and two typical fragment ions, required for their confirmation in single ion monitoring scan mode in animal tissues.     

Conversion of the mycotoxin patulin to the less toxic desoxypatulinic acid by the biocontrol yeast Rhodosporidium kratochvilovae strain LS11

The infection of stored apples by the fungus Penicillium expansum causes the contamination of fruits and fruit-derived products with the mycotoxin patulin, which is a major issue in food safety. Fungal attack can be prevented by beneficial microorganisms, so-called biocontrol agents. Previous time-course thin layer chromatography analyses showed that the aerobic incubation of patulin with the biocontrol yeast Rhodosporidium kratochvilovae strain LS11 leads to the disappearance of the mycotoxin spot and the parallel emergence of two new spots, one of which disappears over time. In this work, we analyzed the biodegradation of patulin effected by LS11 through HPLC. The more stable of the two compounds was purified and characterized by nuclear magnetic resonance as desoxypatulinic acid, whose formation was also quantitated in patulin degradation experiments. After R. kratochvilovae LS11 had been incubated in the presence of (13)C-labeled patulin, label was traced to desoxypatulinic acid, thus proving that this compound derives from the metabolization of patulin by the yeast. Desoxypatulinic acid was much less toxic than patulin to human lymphocytes and, in contrast to patulin, did not react in vitro with the thiol-bearing tripeptide glutathione. The lower toxicity of desoxypatulinic acid is proposed to be a consequence of the hydrolysis of the lactone ring and the loss of functional groups that react with thiol groups. The formation of desoxypatulinic acid from patulin represents a novel biodegradation pathway that is also a detoxification process.     

Water absorption of freeze-dried meat at different water activities: a multianalytical approach using sorption isotherm, differential scanning calorimetry, and nuclear magnetic resonance

Hydration of freeze-dried chicken breast meat was followed in the water activity range of aw=0.12-0.99 by a multianalytical approach comprising of sorption isotherm, differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR). The amount of frozen water and the shape of the T2-relaxogram were evaluated at each water content by DSC and NMR, respectively. Data revealed an agreement between sorption isotherm and DSC experiments about the onset of bulk water (aw=0.83-0.86), and NMR detected mobile water starting at aw=0.75. The origin of the short-transverse relaxation time part of the meat NMR signal was also reinvestigated through deuteration experiments and proposed to arise from protons belonging to plasticized matrix structures. It is proved both by D2O experiments and by gravimetry that the extra protons not contributing to the water content in the NMR experiments are about 6.4% of the total proton NMR CPMG signal of meat.     

Real-time TaqMan polymerase chain reaction detection and quantification of cow DNA in pure water buffalo mozzarella cheese: method validation and its application on commercial samples

Mozzarella cheese obtained from buffalo (Bubalus bubalis) milk is a typical Italian product certificated by means of the European Protected Designation of Origin (PDO). Mozzarella cheese can also be obtained from bovine milk or bovine/buffalo milk mixtures, but in this case, it cannot be sold as PDO product, and its label must report the actual ingredients. However, bovine milk in PDO products was frequently detected in the past, suggesting fraudulent addition or accidental contamination. Several methods based on end-point polymerase chain reaction (PCR) have been profitably applied in a large number of tests to detect the presence of undeclared ingredients, also in dairy products. In the present study we report a real-time PCR method able to quantify bovine milk addition to pure buffalo cheese products. We validated a normalized procedure based on two targets: bovine mitochondrial cytochrome b (cyt b) to detect and quantify the bovine DNA and nuclear growth hormone (GH) gene used as a universal reference marker. With the use of this real-time PCR assay, 64 commercial mozzarella di bufala cheese samples purchased at local supermarkets, dairy shops, or directly from cheese manufacturers were analyzed. The results obtained demonstrate that most of the commercial samples were contaminated with bovine milk. Therefore, this assay could be conveniently employed to carry out routine and accurate controls aimed not only to discourage any fraudulent behavior but also to reduce risks for consumer health.     

Vitamin E composition of walnuts (Juglans regia L.): a 3-year comparative study of different cultivars

The tocopherol and tocotrienol composition of walnuts (Juglans regia L.) was determined for nine cultivars (cvs. Arco, Franquette, Hartley, Lara, Marbot, Mayette, Mellanaise, Parisienne, and Rego). Walnuts were harvested over three consecutive years from two different geographical origins (Bragan?a and Coimbra, Portugal), for a total of 26 samples. The methodology employed was a normal-phase high-performance liquid chromatography coupled to a series arrangement of a diode array detector followed by a fluorescence detector, allowing the simultaneous analysis of all tocopherols and tocotrienols. The analyses showed that all samples presented a similar qualitative profile composed of five compounds: alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, and gamma-tocotrienol. gamma-Tocopherol was the major compound in all samples, ranging from 172.6 to 262.0 mg/kg, followed by alpha- and delta-tocopherols, ranging from 8.7 to 16.6 mg/kg and from 8.2 to 16.9 mg/kg, respectively. Multivariate analysis of the data obtained showed the existence of significant differences in composition among cultivars. These differences were also significant when cultivars were grouped by year of production, showing that besides genetic factors, the vitamin E composition was influenced by environmental factors.     

Liming as an ameliorator of copper toxicity in black oat (Avena strigosa Schreb.)

This study aimed to assess the effect of liming as an ameliorator of copper (Cu) toxicity in black oats (Avena strigosa Schreb.) grown in soil with a sandy texture. The treatments were soil supplementation with increasing doses of lime (0.0, 0.5 and 1.0 g kg^?1) and two of Cu (0 and 50 mg kg^?1). Pre-germinated black oat seeds were sown for 30 days in the soil, subsequently, the plant height, root and shoot dry mass and concentration of total chlorophyll, chlorophylls a and b, carotenoids and total soluble carbohydrates and starch were analyzed. The dose of 50 mg kg^?1 soil Cu caused a toxic effect on black oat plant growth and development, affected the internal distribution of nutrients, decreased the leaf chlorophyll concentration and increased the leaf concentration of total soluble carbohydrates and starch. Increasing lime doses ameliorated the toxic effects of Cu.     

Tracking Anthropogenic Inputs in Barigui River, Brazil Using Biomarkers

The Barigüi River watershed is located in the metropolitan region of Curitiba, Southern Brazil, passing through several neighboring counties. In recent years, due to growth and disorderly occupation along the river, in addition to lack of sanitation, the Barigüi River suffered a very large inflow of untreated domestic wastewater. Current programs to monitor the watershed use traditional physical–chemical parameters. Here, the presence of some molecular tracers found in domestic effluents was investigated, such as caffeine and coprostanol at some selected sites in Barigüi River. Caffeine is highly soluble in water and its presence in water bodies is due to the disposal of untreated sewage. Caffeine is eliminated in the urine (approximately 0.5% to 10% of the consumption). The samples were collected in three campaigns, two in dry weather and another during a week in the rainy season. The results showed that caffeine concentration ranged between zero and 753.5 μg g^?1. Higher values were found on rainy days. Caffeine showed a positive correlation between BOD and nitrate; it also showed a positive correlation with coprostanol, a fecal biomarker. Finally, caffeine has been shown to be a good parameter to determine the contamination by domestic wastewater.     

Nitrogen fertilization of black walnut (Juglans nigra L.) during plantation establishment. Physiology of production

Physiological mechanisms by which nitrogen (N) fertilization affects growth and development in temperate deciduous forest trees are not clearly understood, especially under intensive silvicultural systems. Grafted, Tippecanoe 1 cultivar black walnut (Juglans nigra L.) trees were grown in an intensively managed plantation in west-central Spain and subjected to six, fixed-nutrient-ratio complete fertilizer treatments (defined as 0, 25, 50, 75, 150, and 300 g N tree^?1) delivered via daily fertigation. Leaf chemistry and morphology were evaluated from June to September, and gas exchange was measured in July. Specific leaf mass, leaflet nitrogen (N), and chlorophyll concentrations varied over the course of the growing season, yet consistently increased with increasing fertilization. Net photosynthesis at ambient (A _net) and light-saturated (A _max) conditions increased from the unfertilized control to lowest treatment (25 g N) but did not increase at higher fertilizer rates. Photosynthetic N and chlorophyll use efficiencies decreased with increasing fertilization, but photosynthetic phosphorus and water use efficiencies increased. Transpiration rates and dark respiration were not significantly affected by treatment. Overall, the lowest fertilizer treatment (25 g N) had the greatest photosynthetic efficiency. Interactions between N and other nutrients with increasing fertilizer application suggested potential for nutrient imbalances at high fertilization rates. Our results provide a physiological justification for the use of low-to-moderate fertilization as an efficient strategy to promote black walnut plantation establishment under intensive cultural systems.