Determination of biogenic amines in wine by thin-layer chromatography/densitometry

Biogenic amines are small-molecular-weight organic bases that can be encountered in all fermented foods, including wine. Ingestion of wine containing biogenic amines, and especially histamine, can result in health nuisances. HPLC is the analytical technique most often employed in the determination of biogenic amines in wine but HPLC-based methods are expensive and time-consuming. A new method, based upon amine dansylation and TLC/densitometry, was developed and validated. This allowed for the determination of histamine, tyramine, putrescine and cadaverine in wine at concentrations between 1 and 20mg/L. Analytical performances adequately complied with the needs of routine wine analysis, moreover the method was high-throughput and inexpensive. A simpler, semi-quantitative version of the method, based on visual evaluation of spot intensity, was also developed.     

Performance of blue-yellow screening test for antimicrobial detection in ovine milk

Drug residues in milk are important because of public health and industrial implications. The detection limits of 25 antimicrobial agents were determined by the blue-yellow screening method in ovine milk. For each drug, 8 concentrations were tested on 20 ovine milk samples from individual ewes in midlactation. Detection limits determined by means of logistic regression were below European Union maximum residue limits (EU-MRL) for penicillin G (3 to 4 μg/kg), ceftiofur (96 to 107 μg/kg), framycetin (720 to 781 μg/kg), neomycin (915 to 1,084 μg/kg), and tylosin (44 to 51 μg/kg). Detection limits for ampicillin (5 to 6 μg/kg), cloxacillin (33 to 42 μg/kg), cefoperazone (73 to 82 μg/kg), cefalexin (160 to 202 μg/kg), gentamycin (355 to 382 μg/kg), streptomycin (3,063 to 3,593 μg/kg), tilmicosin (109 to 131 μg/kg), erythromycin (444 to 522 μg/kg), spyramicin (1,106 to 1,346 μg/kg), sulfadimethoxine (101 to 119 μg/kg), sulfathiazole (122 to 151 μg/kg), sulfamethazine (309 to 328 μg/kg), sulfanilamide (1,750 to 2,674 μg/kg), tetracycline (233 to 257 μg/kg), oxytetracycline (398 to 501 μg/kg), doxycycline (323 to 419 μg/kg), chlortetracycline (3,331 to 3,989 μg/kg), danofloxacin (4.7 to 5.5 mg/kg), enrofloxacin (41 to 46 mg/kg), and flumequin (63 to 71 mg/kg) were higher than the EU-MRL. Although the blue-yellow method showed improved sensitivity compared with other tests studied in ovine milk, the performance of screening methods for detecting antimicrobial agents in milk of this species should be improved.     

Fate and transport of faecal contamination microbial indicators, pathogenic protozoa and Campylobacter in the artificially recharged fractured aquifer of Salento, Italy.

The study investigates the fate and transport of microorganisms introduced by artificial groundwater recharge at the Nardò fractured aquifer in Salento, Italy. Microbial indicators of faecal contamination, parasitic protozoa (Giardia and Cryptosporidium) and pathogenic bacteria (Campylobacter spp.), were monitored into injected water and groundwater to test the efficiency of the "natural disinfection" into the fractured aquifer. A remarkable decrease of microbial indicators and pathogens was observed suggesting that pathogens removal or inactivation may be possible during water flow in fractured aquifer. The recently described PNA probe CJE195 (Lehtola et al. 2005) was utilised for the rapid and specific detection of Campylobacter spp. by fluorescence in situ hybridization (FISH) after enrichment. FISH results were consistent with those of traditional cultural method (ISO 17995) applied in parallel: time required for Campylobacter identification was reduced of 4 days.     

Antarctic research bases: local sources of polybrominated diphenyl ether (PBDE) flame retardants

Contemporary studies of chemical contamination in Antarctica commonly focus on remnants of historical local releases or long-range transport of legacy pollutants. To protect the continent's pristine status, the Antarctic Treaty's Protocol on Environmental Protection prohibits importation of persistent organic pollutants. However, some polybrominated diphenyl ether (PBDE) congeners exhibit similar properties. Many modern polymer-containing products, e.g., home/office furnishings and electronics, contain percent levels of flame retardant PBDEs. PBDE concentrations in indoor dust and wastewater sludge from the U.S. McMurdo and New Zealand-operated Scott Antarctic research bases were high. Levels tracked those in sludge and dust from their respective host countries. BDE-209, the major constituent in the commercial deca-PBDE product, was the dominant congener in sludge and dust, as well as aquatic sediments collected near the McMurdo wastewater outfall. The pattern and level of BDE-209 sediment concentrations, in conjunction with its limited environmental mobility, suggest inputs from local sources. PBDE concentrations in fish and invertebrates near the McMurdo outfall rivaled those in urbanized areas of North America and generally decreased with distance. The data indicate that reliance on wastewater maceration alone, as stipulated by the Protocol, may permit entry of substantial amounts of PBDEs and other chemicals to the Antarctic environment.     

Authentication of meat from game and domestic species by SNaPshot minisequencing analysis

The aim of the present study is to develop an assay for the specific identification of meat from Capreolus capreolus, Cervus elaphus, Capra ibex, Rupicapra rupicapra, targeting sequences of the cytochrome b (cyt b) gene of mitochondrial DNA. The assay is also intended to enable differentiation between meat from these wild species as well as Ovis aries, Capra hircus, Bubalus bubalis, Bos taurus and Sus scrofa domestic species.The primers used in the preliminary PCR were designed in well conserved regions upstream and downstream of the diagnosis sites. They successfully amplified a conserved 232 bp region from the cyt b gene of all the species taken into consideration. The sites of diagnosis have been interrogated using a minisequencing reaction and capillary electrophoresis. All the results of the multiplex PER (primer extension reaction) test were confirmed by fragment sequencing. The assay offers the possibility of discriminating nine species at the same time.     

Copper in foods, beverages and waters from South East Spain: influencing factors and daily dietary intake by the Andalusian population

The copper content of 225 food, 49 beverage and twelve potable water samples were determined using atomic absorption spectrometry (AAS). Analyses of NIST and BCR reference materials demonstrated the accuracy of this technique. The highest copper levels were found in dried fruit and legumes, followed by organ meats, molluscs and crustaceans, cephalopods, cereals and sausages, respectively. In cereals, legumes and fruit, copper levels increased significantly with increasing levels of protein and decreasing carbohydrate content (p < 0.001). In meat and meat by-products, copper concentrations found in organ meats were significantly higher (p < 0.01). In fresh fish products, copper levels in shellfish were significantly higher than those measured in fish (p < 0.001). In vegetables, the copper concentrations found in mushrooms were significantly higher (p < 0.005). Mean copper concentrations analysed in cheese were statistically higher than those determined in other dairy products (p < 0.01). In beverages, copper levels determined in rum and juices were significantly higher (p < 0.001). Beverages for which a vegetable component was directly used in their manufacturing process (juices, wines and beers) had statistically higher copper levels when compared with fresh drinks. The daily dietary intake (DDI) of copper in the Andalusian diet was 1979 mug day(-1) per person. Cereals, meat, meat by-products and vegetables are the food categories that are the main source of copper in the daily diet. Taking into account the dietary reference intakes and upper levels (900 and 10, 000 mug Cu day(-1) for healthy adults, respectively), the mean copper DDI found indicate that for most of healthy adult individuals from the area, no adverse effects occur in relation to copper nutrition (deficiency or toxicity). Potable waters supplied 53 mug day(-1), which constitutes on average 0.025% of the maximum tolerable daily intake of this element set by the Joint FAO/WHO Expert Committee.     

Classification of Marsala wines according to their polyphenol, carbohydrate and heavy metal levels using canonical discriminant analysis

Marsala is a popular Sicilian fortified aged wine with ancient tradition. Nowadays Marsala is exported all over the world and is considered one of the most important dessert wines. The aim of this study was to determine the concentration of carbohydrates, polyphenols and heavy metals in different types of Marsala wines and to achieve statistical classifications by stepwise forward canonical discriminant analysis (CDA). The obtained results provided evidence that different types of Marsala were correctly classified according to their phenolic and carbohydrate compositions. In particular, the residual sugars allowed a good discrimination among Marsalas having similar total sugar contents. CDA, performed using heavy metals as independent variables, showed that Superiore Ambra Secco and Vergine Marsalas were not discriminated, whereas a good separation among Fine Oro Dolce, Superiore Riserva and Fine Ambra Secco wines was obtained. Finally, an overall statistical model showed that the variables with the highest discriminant power were: tyrosol, caffeic acid, procyanidin B1, catechin, quercetin, kaempferol, lactose, rhamnose, zinc, copper and lead.     

Cloning, sequencing, and expression of H.a.YNR1 and H.a.YNI1, encoding nitrate and nitrite reductases in the yeast Hansenula anomala

A single Hansenula anomala genomic DNA fragment containing the genes H.a.YNR1 (yeast nitrate reductase) and H.a.YNI1 (yeast nitrite reductase) encoding nitrate and nitrite reductase, respectively, was isolated from a lambda EMBL3 genomic DNA library. As probe, a 3.2 kb DNA fragment isolated from a lambda gt11 H. anomala genomic DNA library screened with antiserum anti-NR from H. anomala was used. H. a.YNR1 and H.a.YNI1 genes are separated by 473 bp and encode putative proteins of 870 and 1077 amino acids, respectively, with great similarity to nitrate and nitrite reductases from other organisms. Northern blot analysis revealed that both genes are highly expressed in nitrate, very low in nitrate plus ammonium, and no expression was detected in ammonium or nitrogen-free media. Levels of nitrate reductase and nitrite reductase were very low or undetectable by Western blot analysis in nitrogen-free and ammonium media, whereas both proteins were present in nitrate and ammonium plus nitrate media. The nucleotide sequence Accession No. is AF123281.     

Qualitative and quantitative analysis of phosphorylated compounds in milk by means of 31P-NMR

31P-NMR has been tested as a technique for determining the phosphorus content of milk samples in both the inorganic form (Pi) and bonded to caseins as phosphoserine (SerP). 31P-NMR made possible the simultaneous determination of Pi and SerP in approximately 1.7 h. The determination is based on measuring the area under the resonance in question and interpolating in a calibration curve prepared using external standards of known phosphorus contents and prepared in the same manner. The concentration area plots obtained were linear in the ranges 162-1993 mg P/l for Pi and 38-40.2 mg P/l for casein SerP. The repeatability of the method was good. When the same milk samples were analysed by both 31P-NMR and the classic colorimetric method the correlation was good. Polyphosphates added to commercial milk were also detected, as diphosphates. The NMR response for diphosphate was linear between 58 and 576 mg P/l and preliminary results indicated that quantitative analysis could be achieved in commercial milk.