Pulsed Electric Field Processing of Red Grapes (cv. Rondinella): Modifications of Phenolic Fraction and Effects on Wine Evolution.

Pulsed electric field (PEF) is a non-thermal technology able to promote color and polyphenols extraction from grape skins. Most of the publications about PEF in winemaking report data concerning international varieties, poorly considering minor cultivars and the medium/long-term effects of the treatment on wine composition during storage. PEF was applied at different specific energies (2, 10, and 20 kJ kg-1) on grapes of the low-color red cv. Rondinella, after crushing-destemming. Pressing yield, the evolution of color, and total phenolic index (TPI) were measured during skin maceration. Moreover, the wines were characterized for basic compositional parameters, color, anthocyanin profile, phenolic composition (glories indices), metal content (Fe, Cr, and Ni), and sensory characters, two and twelve months after the processing, in comparison with untreated samples and pectolytic enzymes (PE). PEF did not affect fermentation evolution, nor did it modify wine basic composition or metal content. Treatments at 10 and 20 kJ kg-1 led to higher color and TPI in wines, in comparison to PE, because of increased content of anthocyanins and tannins. The sensory evaluation confirmed these findings. Modifications remained stable in wines after twelve months. Glories indices and vitisin A content highlighted greater potential stability of wine color in PEF-treated wines.

Analysis of 19 Minerals and Cortisol in Red Deer Hair in Two Different Areas of the Stelvio National Park: A Preliminary Study.

The aim of the study was to perform an investigation on the concentration of 19 minerals and cortisol in red deer (Cervus elaphus) hair, a matrix that is easy to collect with non-invasive and painless sampling, able to represent an integrative values of long-term substance concentrations, and able to give useful information, also when performed on dead animals, given its extreme stability over time. In the study thirty-five animals were included, coming from two different sides of a valley in the Stelvio National Park, where official water analysis had pointed out elevated concentrations of As in one of the two orographic sides. Hair cortisol concentrations were measured using a RIA(Radio Immuno Assay), while minerals were detected using ICP-MS (Inductively Coupled Plasma- Mass Spectrometry). Results showed a negative relationship between cortisol and some mineral concentrations (Li, Co, As, Cd, Cr and Tl) and significant differences in some mineral concentrations between park areas (Al, Co, Cu, Cd and Ni). As, Cr and cortisol differences approached statistical significance. This preliminary study represents a step forward in the study of wildlife allostatic load and a valid method for applications in wildlife management programs, in environmental studies and in public health programs.

A cotton thread fluidic device with a wall-jet pencil-drawn paper based dual electrode detector.

A simple and sensitive device is presented based on the use of pencil-drawn paper based electrochemical detector placed at the end of a cotton thread fluidic channel in wall-jet configuration. This innovative and fast responding electroanalytical system can be adopted for both single and dual electrode electrochemical detection, this last achieved by applying two different potentials at two independent working electrodes drawn on the opposite faces of the paper based detector. Its performance was preliminarily optimized by adopting hexacyanoferrate(II) as probe species undergoing reversible electrochemical processes. These devices were then used for the single electrode detection of ascorbic acid in aqueous samples and the dual electrode detection of orthodiphenols in extra virgin olive oils (EVOOs). In fact, these devices enable hydrophilic orthodiphenols, typically present in EVOOs (extracted by a 80:20% v/v acetonitrile/water mixture), to be discriminated from hydrophilic monophenols instead present in almost all vegetable oils. Flow-injections runs were conducted by using a 0.01 M H2SO4 + 0.5 KCl running electrolyte allowing the rapid and selective detection of hydrophilic orthodiphenols with satisfactory sensitivity and a low enough detection limit (2 µM). Different real samples of EVOOs and sunflower oils were analyzed. Abundant enough contents of orthidiphenols were found in EVOO samples, while no trace of these antioxidants was found in sunflower oils.

Digitally Controlled Procedure for Assembling Fully Drawn Paper-Based Electroanalytical Platforms.

A simple, reliable, and low-cost fabrication method is proposed here for assembling paper-based electrochemical devices (PEDs) using a commercial desktop digitally controlled plotter/cutter, together with ordinary writing tools. Permanent markers (tips of 1 mm) were used to create effective hydrophobic barriers on paper, while micromechanical pencils (mounting 4B graphite leads, 0.5 mm in diameter) were adopted for automatically drawn precise reference, counter, and working carbon electrodes. Fabrication parameters, such as writing pressure and speed, were first optimized, and the electrochemical performance of these devices was then evaluated by using potassium hexacyanoferrate(II) as redox probe. The good interdevice reproducibility (4.8%) displayed by the relevant voltammetric responses confirmed that this strategy can be profitably adopted to easily assemble paper-based electrochemical devices in a highly flexible manner. The simplicity of the instrumentation used and the low cost of each single device (about $0.04), together with the speed of fabrication (about 2 min), are other important features of the proposed strategy. Finally, to confirm the effectiveness of this prototyping method for the analysis of real samples and rapid controls, PEDs assembled by this simple approach were successfully exploited for the analysis of vitamin B6 in food supplements.

Survival-Related Autophagic Activity Versus Procalcific Death in Cultured Aortic Valve Interstitial Cells Treated With Critical Normophosphatemic-Like Phosphate Concentrations.

Valve dystrophic calcification is a common disorder affecting normophosphatemic subjects. Here, cultured aortic valve interstitial cells (AVICs) were treated 3 to 28 days with phosphate (Pi) concentrations spanning the normal range in humans (0.8, 1.3, and 2.0 mM) alone or supplemented with proinflammatory stimuli to assess possible priming of dystrophic-like calcification. Compared with controls, spectrophotometric analyses revealed marked increases in calcium amounts and alkaline phosphatase activity for 2.0-mM-Pi-containing cultures, with enhancing by proinflammatory mediators. Ultrastructurally, AVICs treated with low/middle Pi concentrations showed an enormous endoplasmic reticulum (ER) enclosing organelle debris, so apparently executing a survival-related atypical macroautophagocytosis, consistently with ultracytochemical demonstration of ER-associated acid phosphatase activity and decreases in autophagosomes and immunodetectable MAP1LC3. In contrast, AVICs cultured at 2.0-mM Pi underwent mineralization due to intracellular release and peripheral layering of phospholipid-rich material acting as hydroxyapatite nucleator, as revealed by Cuprolinic Blue and von Kossa ultracytochemical reactions. Lack of immunoblotted caspase-3 cleaved form indicated apoptosis absence for all cultures. In conclusion, fates of cultured AVICs were crucially driven by Pi concentration, suggesting that serum Pi levels just below the upper limit of normophosphatemia in humans may represent a critical watershed between macroautophagy-associated cell restoring and procalcific cell death.

A paper-based platform with a pencil-drawn dual amperometric detector for the rapid quantification of ortho-diphenols in extravirgin olive oil.

A simple, sensitive and fast responding device is described for the discrimination of hydrophilic ortho-diphenols, whose presence in abundant enough amounts is typical for extra virgin olive oils (EVOOs), from hydrophilic mono-phenols instead present in almost all vegetable oils. It consists of a dual electrode detector pencil-drawn at the end of a paper microfluidic channel, defined by hydrophobic barriers, where samples of these antioxidants, extracted from vegetable oils by a 80:20% v/v acetonitrile/water mixture, were applied. Thin-layer chromatographic runs conducted by using a 0.01 M H2SO4 + 1 M KCl running buffer allowed the selective detection of hydrophilic ortho-diphenols by profiting from the fact that they undergo reversible oxidation at less positive potentials than those required by monophenols for displaying their irreversible anodic process. On this basis, a potential for the oxidation of hydrophilic ortho-diphenols was applied to the upstream pencil-drawn electrode (W1) (at which a minor fraction of mono-phenols was also oxidized), while a potential for the reverse process involving the sole product (ortho-quinones) of the reversible oxidation of ortho-diphenols was imposed at the downstream pencil-drawn working electrode (W2). Thus, cathodic peak currents linearly dependent on analyte concentrations could be recorded at W2 which led to a satisfactory detection limit (8 µM, equivalent to 1.23 mg/L) even when working electrodes W1 and W2 with same dimensions were employed. Improved sensitivities and lower detection limits were achieved by increasing the dimensions of W2 with respect to W1, thanks to the improvement of the collection efficiency. Throughout this investigation, hydroxytyrosol (HTy) and tyrosol (Ty) were adopted as models of ortho-diphenols and mono-phenols, respectively, in view of their abundant presence in EVOOs. Real samples of EVOO from different production companies, of a simple olive oil and of a sunflower oil were analyzed. Different hydrophilic ortho-diphenol contents were found in EVOO samples (up to 40.8 mg/kg), while only a negligible amount turned out to be present in simple olive oil. No trace of these antioxidants were instead found in sunflower oil, as expected. All concentrations found were in good agreement with those detected by a more frequently employed spectrophotometric method used for the sake of comparison.

Seasonal changes in technological and nutritional quality of Mytilus galloprovincialis from suspended culture in the Gulf of Trieste (North Adriatic Sea).

Nutritional quality parameters, microbiological and technological quality indicators (condition index, meat yield and water-holding capacity) of blue mussel, Mytilus galloprovincialis, reared in the North Adriatic Sea were characterised at monthly intervals over a 1 year period. Contents of protein (7.5-11.6 g/100 g), lipid (1.0-2.2 g/100 g) and ash (2.2-3.3 g/100 g) varied significantly accordingly to condition index (6-15%). n-3 PUFAs were the predominant fatty acids (38.7-45.9% of fatty acids) and docosahesaenoic and eicosapentaenoic acids were the most abundant (167 and 93.3 mg/100 g, respectively). Glycine, glutamic and aspartic acids accounted for 40% of total amino acids. All samples exhibited limited concentrations of Cr, Mn, Ni, Cu and Zn, as well as Na. M. galloprovincialis from the North Adriatic Sea showed the highest technological and nutritional quality, considering also the inter-annual variability, in late spring, which corresponds to the period immediately before gamete release.

Ultrastructural and spectrophotometric study on the effects of putative triggers on aortic valve interstitial cells in in vitro models simulating metastatic calcification.

Metastatic calcification of cardiac valves is a common complication in patients affected by chronic renal failure. In this study, primary bovine aortic valve interstitial cells (AVICs) were subjected to pro-calcific treatments consisting in cell stimulation with (i) elevated inorganic phosphate (Pi = 3 mM), to simulate hyperphosphatemic conditions; (ii) bacterial endotoxin lipopolysaccharide (LPS), simulating direct effects by microbial agents; and (iii) conditioned media (CM) derived from cultures of either LPS-stimulated heterogenic macrophages (commercial murine RAW264.7 cells) or LPS-stimulated fresh allogenic monocytes/macrophages (bCM), simulating consequent inflammatory responses, alone or combined. Compared to control cultures, spectrophotometric assays revealed shared treatment-dependent higher values of both calcium amounts and alkaline phosphatase activity for cultures involving the presence of elevated Pi. Ultrastructurally, shared peculiar pro-calcific degeneration patterns were exhibited by AVICs from these latter cultures irrespectively of the additional treatments. Disappearance of all cytomembranes and concurrent formation of material showing positivity to Cuprolinic Blue and co-localizing with silver precipitation were followed by the outcropping of such a material, which transformed in layers outlining the dead cells. Subsequent budding of these layers resulted in the formation of bubbling bodies and concentrically laminated calcospherulae mirroring those in actual soft tissue calcification. In conclusion, the in vitro models employed appear to be reliable tools for simulating metastatic calcification and indicate that hyperphosphatemic-like conditions could trigger valve calcification per se, with LPS and allogenic macrophage-derived secretory products acting as possible calcific enhancers via inflammatory responses.

Pro-calcific responses by aortic valve interstitial cells in a novel in vitro model simulating dystrophic calcification.

Etiopathogenetic mechanisms in calcific aortic valve stenosis are still poorly understood despite this being the third major cause of heart disease in western world. In prior in vitro cultures simulating metastatic calcification, pro-calcific effects on aortic valve interstitial cells (AVICs) resulted by adding bacterial endotoxin lipopolysaccharide (LPS) at high inorganic phosphate (Pi) levels. Here we accomplished improved in vitro models simulating either metastatic (Pi = 2.6 mM) or dystrophic calcification (Pi = 1.3 mM), in which LPS-stimulated bovine AVICs underwent extra-stimulation with macrophage-cytokine-containing media derived from parallel cultures of allogeneic monocyte/macrophages in turn stimulated with LPS. In dystrophic calcification-like cultures, lower calcium amount was spectrometrically assessed with parallel reduced alkaline phosphatase activity with respect to metastatic calcification-like cultures, with an about three-fold slower progression of mineralization. Hydroxyapatite crystal precipitation was ultrastructurally found to correlate with AVIC degeneration processes culminating with the formation of phthalocyanin-positive lipidic layers (PPLs) at the surface of cells and cell-derived matrix-vesicle-like bodies, acting as calcium nucleators according to a pattern mirroring those we had previously found in in vivo conditions. In conclusion, an in vitro model has been developed enabling reliable simulations of the effects exerted on AVICs by putatively pro- or anti-calcific agents.

Alcohol reduces MMP-2 in humans and isolated smooth muscle cells.

Alcoholic beverages are known to exert a protective effect on atherosclerotic disease. This study aimed to assess the in vivo and in vitro effects of alcohol on matrix metalloproteinase (MMP) -2 and -9, known to determine atherosclerosis progression. Eighteen healthy volunteers, regular drinkers (two standard alcohol servings/day, on average) at first examination (baseline) were asked to abstain from any alcoholic beverage for one week (abstention), and then to assume two standard alcohol servings of beer daily for 1 week (re-exposure). Activity of MMP-2 and -9, total antioxidant activity (AOA), glutathione (GSH) plasma levels were carried out at baseline, at the end of abstention, and after 1 week of re-exposure. To validate the in vivo results, MMP-2 activity and expression, AOA, and GSH, were determined in human smooth muscle cells treated for 96 h with increasing concentrations (12.5-100 mM) of ethanol. MMP-2, but not MMP-9 plasma activity was higher at abstention than at baseline or re-exposure (P<.001 and P< or =.005, respectively). Changes in AOA and GSH throughout the study were not significant. No correlation was found between MMPs and antioxidant activity. In vitro, ethanol at 25 mM reduced by around 10% MMP-2 activity (P=.003) in smooth muscle cells, whereas MMP-2 expression, AOA, and GSH were unaffected. Alcohol reduces MMP-2 plasma activity in healthy humans and in isolated vascular smooth muscle cells. This in vitro reduction is unrelated to MMP-2 expression in vascular cells or to antioxidant levels changes.

Lipoxygenase distribution in coffee (Coffea arabica L.) berries.

In this paper lipoxygenase (LOX) presence was investigated in coffee berries to determine its involvement in lipid degradative metabolism of plants grown in organic and conventional cultivations. An immunochemical analysis has evidenced a ca. 80 kDa protein, cross-reacting with an anti-LOX antibody, only in the pulp fraction of berries obtained from plants of both cultivations. LOX activity in this fraction could be monitored either as conjugated diene formation or reaction products (determined by HPLC) and was mainly associated with a heavy membrane fraction (HMF, enriched in tonoplast, endoplasmic reticulum, plasma membrane, and mitochondria) and a light membrane fraction (LMF, enriched in plasma membrane and endoplasmic reticulum, with low levels of tonoplast and mitochondria). The LOX activity of LMF from berries of both cultivations showed an optimum at pH 8.0. The HMF exhibited a different activity peak in samples from conventional (pH 8.0) and organic (pH 5.5) cultures, suggesting the presence of different isoenzymes. These findings were also confirmed by variation of the ratio of 9- and 13-hydroperoxides in organic (1:1) and conventional cultivations (1:10), indicating that the organic one was subjected to an oxidative stress in the coffee pulp fraction leading to the expression of an acidic LOX. Such de novo synthesized LOX activity could be responsible for the production of secondary metabolites, which may interfere with the organoleptic profile of coffee.

Selenium supplementation prevents the increase in atherogenic electronegative LDL (LDL minus) in the postprandial phase.

Evidence is accumulating that postprandial phenomena play a role in atherogenesis. Dietary lipid hydroperoxides that escape from the gastrointestinal barrier can be incorporated into plasma lipoproteins, leading to a modified form of LDL (LDL minus). The present human study was designed to investigate the effect of selenium supplementation on the formation of LDL minus in the postprandial phase. Fourteen healthy subjects ate the same test meal, high in lipid hydroperoxides, at baseline and after 10-day selenium supplementation (110 microg/day). Plasma selenium, LDL minus, LDL resistance to oxidative modification, plasma antioxidants (ascorbic acid, GSH and GPx activity) and MDA were measured in preprandial (time 0) and postprandial (3h) phases. Supplementation did not induce changes in the concentration of selenium in fasting plasma, but, at the same time, it induced a significant decrease in preprandial plasma GPx activity and inhibited the meal-induced increase in GPx activity. Selenium supplementation fully prevented the meal-induced increase in both LDL minus level and LDL susceptibility to oxidation. This study demonstrated the efficacy of selenium in preventing postprandial oxidative stress. The results, obtained on subjects adequately supplied with selenium, suggest that a non-limiting selenium availability counteracts the postprandial formation of the atherogenic form of LDL and provide a rationale for the epidemiological evidence of the inverse correlation between selenium intake and the incidence of chronic and degenerative diseases.

Analysis of metals and EOX in sludge from municipal wastewater treatment plants: a case study.

The monitoring of extractable organic halogen (EOX) and heavy metal contents in sludge coming from 10 different municipal wastewater treatment plants (MWWTP) located in Friuli Venezia Giulia (Italy) is reported. In this work, sludge samples drawn from sludge treatment units have been digested and analyzed by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) for metal evaluation. Samples were also extracted and analyzed by microcoulometric titrations, following modified DIN 38414 T17 standard, for EOX analysis. Analytical results showed a slight enrichment of the contents of certain metals (Cd< 2mg/kg, Cr< 51.5mg/kg, Cu<105.8 mg/kg, Hg<1.4 mg/kg, Ni<35.9 mg/kg, Pb<58.7 mg/kg, Zn<410.1 mg/kg, Ba<317.1 mg/kg, Co<1 mg/kg, Mo< 5 mg/kg, Mn<106.7 mg/kg), so almost all of the sludge would be suitable for agricultural use following Italian and European regulations. The evaluation of EOX was carried out by using hexane and ethyl acetate as extraction solvents, and a measurable organic halogen content (ranging from 0.31 to 39.5 mg Cl/kg DM) was clearly detected in the sludge. The lowest concentrations of EOX were found in sludge coming from the smallest MWWTPs, which is to be considered more suitable for agricultural use. Additionally, analytical assays on composts, peat and soils were performed to compare EOX concentrations between these matrices and sludge.

Evaluation of chlorinated by-products in drinking waters of Central Friuli (Italy).

Drinkable water supplied by aqueducts undergoes preliminar potabilization which, in Italy, is mainly accomplished by chlorine addition. The bactericidal action involved in this process is always accompanied by chlorination and oxidation of organic species (mainly humic and fulvic acids) naturally present in treated waters, so that many disinfection by-products (DBPs) are formed, such as trihalomethanes (THMs) and halo-acetic acids (HAA), which can represent a chemical risk for public health. The aim of this study was the monitoring of DBPs in drinking water disinfected by chlorination, supplied by four different aqueducts of Central Friuli (Italy). DBP evaluations were performed in water samples consisting of both input and output of disinfection plants. The results of analytical determinations were worked out to provide the THM and HAA parameters for disinfected waters, while in feeding waters the following different conventional parameters were adopted: (i) trihalomethanes formation potential (THMFP), (ii) halo-acetic acids formation potential (HAAFP) and (iii) UV absorbance at 254 nm (UV254). The quite moderate content of chlorinated products found in all samples considered highlighted the excellent quality of potabilized waters available in Central Friuli. Moreover, our results confirmed that the majority of DBPs formed when chlorine is used for water disinfection consists of THMs, while chlorites and chlorates prevailed when potabilization is accomplished by using chlorine dioxide. Finally, simple UV254 monitoring turned out to be a profitable approach for the determination of chlorinated by-products only when THMs prevail among DBPs.

Biochemical and immunochemical evidences for the presence of lipoxygenase in plant mitochondria.

In this paper, both biochemical and immunochemical evidence for the presence of lipoxygenase (LOX) in plant mitochondria is presented. Highly purified pea (Pisum sativum L., cv. Alaska) mitochondria show LOX activity, evaluated as conjugated diene formation, oxygen consumption, and hydroperoxide formation. Both 9- and 13-hydroperoxy-octadecadienoic acids are produced by the oxidation of linoleic acid. LOX activity is particularly evident in swollen mitochondria; it is inhibited by nordihydroguaiaretic acid, a pea anti-LOX B antibody, and has two pH optima (6.0 and 7.5). A mitochondrial protein of approximately 97 kDa cross-reacts with a pea seed anti-LOX B antibody. This reaction is detectable in both soluble (matrix fraction) and membrane-bound (submitochondrial particles) proteins. Considering that pea mitochondria were extracted from actively growing stems that were differentiating tube elements, it is suggested that the presence of LOX in these organelles may be related to their degradation linked to xylem differentiation.

ICP-MS determination of toxic-metal release from pumping systems for food processing.

The release of heavy metals from uncovered and nickel-covered brass pumps has been evaluated by ICP-MS analysis in both simple ultrapure water and 3% acetic acid solution (mimic of neutral and acid edible liquids, respectively), following a procedure similar to that recommended by the National Sanitation Foundation (NSF) International, Test Procedure P203. The results found highlight that the main release regards zinc, copper and lead, i.e. the three major metals present in brass alloys. The first contact of brass surfaces with the extraction solvent leads to an extensive Pb release which is comparable with that observed for Cu and Zn. Subsequent washings reduce markedly the Pb release, thus rising in evidence a progressive surface passivation. In particular, the Pb release found after four repeated washings turns out to approach the limit set by both Italian and USA governments for liquids used for food purposes when determined in neutral media, while it remains quite higher when evaluated in acid media. Release analyses conducted on nickel-covered brass pumps point out that the Niploy nickel coating process is very effective for brass surface protection, in that the Pb release is reduced of about three orders of magnitude, but a Ni release exceeding the relevant permitted level is in this case observed.