Evolution in the Bottling of Cabernet Sauvignon Wines Macerated with Their Own Toasted Vine-Shoots.

This work studies, for the first time, the effect of the use of Cabernet Sauvignon vine-shoots as an enological additive (called "Shoot Enological Granule", SEG) in wines of the same variety. SEGs were added in two doses (12 and 24 g/L) at the end of malolactic fermentation, and after that, wines were bottled for six months. The phenolic and volatile composition and sensory profiles of wines were analyzed at bottling and after six months. The results showed a decrease in the total content of phenolic compounds with bottle time; however, stilbenes─specifically trans-resveratrol─were maintained at significant levels in SEG wines. In contrast, the total content of volatile compounds, mainly esters, increased with bottle aging. Finally, in terms of sensory profile, SEG wines showed a clear differentiation between the descriptors and the control, with more-integrated aromas after bottle time with more toasted, nutty vanilla notes, as well as silkier and less bitter tannins, compared to the control.

Toasted Vine Shoots as an Alternative Enological Tool. Impact on the Sensory Profile of Tempranillo Wines during Bottle Aging.

The use of toasted vine shoots (SEGs) as an enological tool is a new practice that seeks to improve wines by differentiating between them and encouraging sustainable wine production. The sensorial impact during bottle aging of wines treated with SEGs is a key factor to consider. This paper studies the influence of SEGs on Tempranillo wines treated with their own SEGs in two different doses (12 and 24 g/L) at two differences moments (during alcoholic fermentation and after malolactic fermentation) throughout 1 year of bottle aging. The results indicate that addition moment is the factor that most affects the evolution of sensorial descriptors. The greatest evolution in the wines was observed during the first 4 months, wherein improved integration of the notes related to addition of SEGs occurred. A reduction in the perception of dryness and bitterness was observed in the treated wines, therefore, SEGs could be considered accelerators to eliminate these initial sensations from wines.

Pruned vine-shoots as a new enological additive to differentiate the chemical profile of wines.

For this study, Tempranillo wines were made by adding their own toasted vine-shoots (SEGs, "Shoot- Enological - Granule"). The SEGs were added in two doses (12 and 24 g/L) at three different times (before alcoholic fermentation, in the middle of alcoholic fermentation, and after fermentations) and phenolic, volatile, and mineral composition were analysed. Results showed a decrease in the total content of phenolic compounds but stilbenes, specifically trans-resveratrol, increased in all wines macerated with SEGs, as did total anthocyanins when these additives were added in the middle of fermentation. Furthermore, the ratios related to glycosylated monomeric anthocyanins were significantly higher in wines treated with SEGs. The use of SEGs did not affect the total content of volatile compounds. However, changes in terms of individual compounds resulted in an odorant series associated with SEGs, named "sweet woody", formed by compounds such as ethyl vanillate, ethyl cinnamate, and vanillin. Finally, the mineral composition of the wines was not affected using SEGs, whereby potassium was the most abundant in all the wines.

Spraying ozonated water on Bobal grapevines: Effect on grape quality.

Ozone is a powerful oxidant that is increasingly used as sanitizing agent in the wine industry and even in the vineyard to control grapevine diseases. In this study, we evaluated the effect on grape enological quality of ozonated water spraying treatments carried out in Bobal grapevines during two consecutive harvest seasons. In the first season, ozonated water was applied once during the ripening period on grapevines trained on the traditional gobelet system (S1). In the second season, ozonated water was applied three times between the fruit set and harvest on grapevines grown on a vertical trellis system (S2). Grape quality on harvest day was evaluated through several enological and chromatic parameters, the phenolic maturity, the Varietal Aroma Potential Index (IPAv) and the phenolic and volatile composition. The S1 treatment had a positive effect on the technological maturity, the chromatic parameters, the seed maturity and the content of glycosylated aroma precursors, phenolic compounds and free terpenoids of grapes. The S2 treatment also improved the technological maturity and the content of total anthocyanins (pH 1.0) and free terpenoids, but had a negative impact on the chromatic parameters, the anthocyanin extractability and the content of glycosylated aroma precursors and phenolic compounds. Therefore, ozonated water sprayed on Bobal grapevines affected the quality of grapes, but the effect seemed to depend on the number of applications.

Moscatel vine-shoot extracts as a grapevine biostimulant to enhance wine quality.

Non-aromatic vine-shoot extracts (Airén) has been recently proposed as "viticultural biostimulants" when applied to grapevine. In this paper, the application of extracts from non-toasted (MVS) and toasted (MVSToasted) vine-shoots from the well-known aromatic variety such Moscatel were applied on Airén grapevine leaves, observing an increased for grape yield and wines with a lower alcohol degree. All wines at the end of the alcoholic fermentation were characterized by their fruity and floral descriptors, especially MVS wines; and 4 months later, MSV and MVSToasted wines surprised by their higher spicy notes, which correspond with the highest OAVs values for compounds such as norisoprenoids (ß-damascenone), vanillin derivatives (vanillin, acetovanillone) and volatile phenols (guaiacol, syringol), compared to control wine. Wine phenolic composition was affected positively over all by MVS in case of phenolic acids. These results confirm that Moscatel vine-shoot extracts foliar application into Airén non-aromatic grapevines produce an interesting enhance on wine quality.

Effect of toasting on non-volatile and volatile vine-shoots low molecular weight phenolic compounds.

Low molecular weight phenolic compounds (LMWPC), including non-volatile and volatile, of Airén and Moscatel vine-shoot cultivars waste submitted to different toasting conditions (light, 180°/15min; medium, 180°/30min; high 180°/45min) were studied in order to exploit them with oenological purposes. The LMWPC differences were mainly due to the toasting times rather than vine-shoot variety. In non-volatile LMWPC fraction, flavanols and almost all phenolic acids decreased by toasting. The presence of trans-resveratrol has a special relevance at light toasting: 14 times more concentrated in Airén and 6 times in Moscatel vine-shoots, than their respective non-toasted samples. The volatile LMWPC showed a significant increment with toasting, being vanillin the one with the highest difference respect to non-toasted samples at high conditions: more than 15 times in Airén and 11 in Moscatel. Although toasting reduced some LMWPC, particular characteristics of these vine-shoots must be taken into account when considering its future use.

Effect of vine foliar treatments on the varietal aroma of Monastrell wines.

The effects of four vine treatments, comprising the application of eugenol and guaiacol (individually or as a mixture) or whiskey lactones on the concentration of glycosidically bound aroma precursors, determined as glycosyl glucose content by HPLC-IR, in Monastrell grapes and their wines were studied. The impact of treatments on the free varietal wine aroma determined by SBSE-GC-MS and descriptive analysis after alcoholic and malolactic fermentations and six months of ageing were also determined. A synergistic effect was observed between the eugenol and guaiacol on the glycosidically bound aroma precursor fraction. The rate of release of such aroma precursors was time and treatment dependent. The impact on wines varietal aroma at the end of the alcoholic fermentation was reduced by treatments, whereas the opposite effect was observed in the following samplings. At a sensory level, the wood/oak notes were appreciated in all wines; however, the typicity of the Monastrell variety was especially enhanced at the end of the malolactic fermentation, in the wines from whiskey lactone treatment.

Local anesthetics inhibit Ca-ATPase in masticatory muscles.

Local anesthetics have myotoxic effects and inhibit Ca-ATPase activity and Ca transport in skeletal muscles. Such effects have not been fully elucidated in masticatory muscles. We tested the hypothesis that local anesthetics increase myoplasmic calcium in masticatory muscles by inhibiting Ca-ATPase at a concentration similar to that of dental cartridges. The effects of lidocaine and bupivacaine on Ca-ATPase from rabbit masseter and medial pterygoid muscles were tested with radioisotopic and colorimetric methods. Bupivacaine had an action similar to that of lidocaine on Ca-ATPase activity, but less effect on calcium transport. The pre-exposure of the membranes to the anesthetics enhanced the Ca-ATPase activity in the absence of calcium ionophore, supporting their permeabilizing effect. The results demonstrate that amide-type anesthetics do not inhibit calcium binding, but do reduce calcium transport and enzyme phosphorylation by ATP, and suggest that the myoplasmic calcium increase induced by lidocaine and bupivacaine might promote masticatory muscle contraction and eventual rigidity.

Characterization of a functional NTPDase in the endoplasmic reticulum of rat submandibular salivary gland.

Nucleotidase activity and Ca-uptake were characterized in endoplasmic reticulum (ER) enriched rat submandibular gland (SMG) microsomal preparations. (i) Ca-uptake had characteristics of an ER Ca-ATPase. (ii) Nucleotidase activity was equally stimulated by calcium, magnesium and manganese, but with different Km values. (iii) Specific inhibitors of P-type Ca-ATPases were ineffective on nucleotidase activity, demonstrating that this activity was not related to calcium uptake and did not correspond to classical Ca(2+) pumps. (iv) ATP and UTP were more efficient substrates, whereas ADP and UDP were hydrolyzed at significantly slower rate. (v) Nucleotidase activity was sensitive to mild detergent solubilization and insensitive to ionophore addition. (vi) Nucleotidase activity was strongly inhibited by suramin, a nucleoside triphosphate diphosphohydrolase (NTPDase) inhibitor. (vii) Nucleotidase activity exponentially diminished as function of time. All these observations are consistent with a NTPDase identity. The presence of a NTPDase was demonstrated by immunohistochemistry in rat SMG. Immunoreactivity was stronger in ductal cells than in mucous and serous acini. Although this enzyme was observed in the plasma membrane, colocalization with the ER marker calnexin revealed a specific subcellular localization in this organelle of all three types of cell. The putative function of this NTPDase activity in salivary glands is discussed.

A new approach to saffron aroma.

The aroma of saffron has received much attention from scientists in recent years, not only for the compounds that make it up, but also for its glycosidic precursors. Despite it all, the volatile generation mechanisms of the spice are almost completely unknown. Only the generation of safranal, the major compound, from picrocrocin has been established. The great effort carried out to detect and identify the volatile compounds of saffron has not been enough to clarify which compounds are responsible for saffron aroma. In general, scientists has devoted little attention to the sample analyzed, taking for granted that all saffron is the same, something that makes it difficult to establish a comparison between the results obtained by the different authors, to the point that saffron aroma has not been defined yet. It must be clarified whether saffron aroma is what the consumer perceives via nasally when a container of the spice is uncovered, or whether on the contrary, it is the aroma conferred to food, normally after a thermal cooking process, and perceived retronasally. After an extensive bibliographic research, simple assays are suggested to understand what could be considered as saffron aroma, results that may help to delimit the research for future studies.

In silico kinetic study of the glucose transporter.

Glucose transport in plasma membranes is the prototypic example of facilitated diffusion through biological membranes, and transport in erythrocytes is the most widely studied. One of the oldest and simplest models describing the kinetics of the transport reaction is that of alternating conformers, schematized in a cycle of four partial reactions where glucose binds and dissociates at two opposite steps, and the transporter undergoes transconformations at the other two opposite steps. The transport kinetics is entirely defined by the forward and backward rate constants of the partial reactions and the glucose and transporter concentrations at each side of the membrane, related by the law of mass action. We studied, in silico, the effect of modifications of the variables on the transient kinetics of the transport reaction. The simulations took into account thermodynamic constraints and provided results regarding initial velocities of transport, maximal velocities in different conditions, apparent influx and efflux affinities, and the turnover number of the transporter. The results are in the range of those experimentally reported. Maximal initial velocities are obtained when the affinities of the ligand for the transporter are the same at the extra- and intracellular binding sites and when the equilibrium constants of the transconformation steps are equal among them and equal to 1, independently of the obvious effect of the increase of the rate constant values. The results are well adjusted to Michaelis-Menten kinetics. A larger initial velocity for efflux than for uptake described in human erythrocytes is demonstrated in a model with the same dissociation constants at the outer and inner sites of the membrane. The larger velocities observed for uptake and efflux when transport occurs towards a glucose-containing trans side can also be reproduced with the alternating conformer model, depending on how transport velocities are measured.

Analysis of wine primary aroma compounds by stir bar sorptive extraction.

Due to the great importance of some primary aroma compounds on wine quality, these compounds which includes terpenes, C(13)-norisoprenoids and C(6) compounds, have been analyzed by stir bar sorptive extraction (SBSE) followed by a thermal desorption-gas chromatography-mass spectrometry analysis. The stir bar sorptive extraction method was optimized in terms of temperature, time, pH and NaCl addition. The best SBSE sorption kinetics for the target analytes were obtained after submitting the solutions to 60 degrees C during 90min. The addition of sodium chloride did not enhance the volatile extraction. The method proposed showed good linearity over the concentration range tested, with correlation coefficients higher than 0.98 for all the analytes. The reproducibility and repeatability of the method was estimated between 0.22 and 9.11%. The detection and quantification limits of all analytes were lower than their respective olfactory threshold values. The application of this SBSE method revealed that monovarietal white wines were clearly separated by two canonic discriminating functions when grape varieties were used as differentiating variable, the first of which explained 98.4% of the variance. The compounds which contributed most to the differentiation were limonene, linalool, nerolidol and 1-hexanol.

Stir bar sorptive extraction for the determination of volatile compounds in oak-aged wines.

Stir bar sorptive extraction (SBSE) followed by a thermal desorption-gas chromatography-mass spectrometry analysis has been optimized for the determination of 13 of the most important oak volatiles in wine samples, all in a single run. The stir bar sorptive extraction method was optimized in terms of temperature, time, pH and NaCl addition, and the best results were obtained after stirring the wine sample with the polydimethylsiloxane stir bar during 90min at room temperature. The addition of sodium chloride did not enhance the volatile extraction. The method proposed showed good linearity over the concentration range tested, with correlation coefficients ranging from 0.92 to 0.99 for all the analytes. The reproducibility and repeatability of the method was estimated between 0.11 and 5.45%. The detection and quantification limits of all analytes were lower than their respective olfactory threshold values, and, most importantly, no artifacts have been observed during the analysis as described in most extractions using other current methodologies.

Screening method for the detection of artificial colours in saffron using derivative UV-Vis spectrometry after precipitation of crocetin.

A screening method for the detection of artificial colours (naphthol yellow, tartrazine, quinoline yellow, Sunset yellow, Allura red, amaranth, azorubine, Ponceau 4R and Red 2G) in saffron is described. The method involves removal of crocins by precipitation of crocetin (pH 0.1, 90 degrees C) before adsorption of the artificial colours on polyamide SPE cartridges (pH 2). After washing with methanol, acetone and methanol, elution was done with a methanol:ammonia solution (95:5 v/v), and detection was performed by derivative spectrometry. Sample pretreatment changes the UV-Vis saffron extract profile in such a way that second derivative spectra can be used to identify the presence of added colours. Erythrosine, which was found to be pH dependent, could not be detected under the above conditions. The lowest detectable amount for each colour was strongly dependent on chemical structure. The recovery of carminic acid was very low possibly due to irreversible retention on the polyamide. This procedure can replace the current ISO TLC method (2003) and be used alternatively or in combination with HPLC procedures adopted in the same standard.

Characteristics of the sarcoplasmic reticulum Ca2+-dependent ATPase from masticatory muscles.

We compared the sarcoplasmic reticulum (SR) Ca-ATPase from masseter (M) and medial pterygoid (MP) muscles with that from fast muscles (FM) to examine whether its calcium transport capability and enzymatic activity are different. SR vesicles from FM, M, and MP muscles were obtained according to Champeil et al.(1985). Assays for characterization of the enzyme properties were performed. The results showed similar optimal conditions for the Ca-ATPase activity and calcium transport in M, MP, and FM. However, the maximal values of calcium transport, Ca-ATPase activity, and K(i) for thapsigargin were significantly lower in the masticatory muscles. These findings are likely related to different Ca-ATPase isoforms. Since the local anesthetics used in dentistry inhibit Ca-ATPase and calcium transport in FM, it will be important for the effects of these drugs on the Ca-ATPase of masticatory muscles to be assessed.

Stir bar sorptive extraction for the analysis of wine cork taint.

A magnetic stir bar with a polydimethylsiloxane coating was used to absorb 2,4,6-trichloroanisole, 2,3,4,5-tetrachloroanisole, pentachloroanisole and their respective phenols from synthetic and real wine samples. The stir bar sorptive extraction method was optimised to obtain the best extraction conditions in terms of temperature, time, pH and NaCl addition. The stir bar was desorbed in a thermal desorption system coupled to a gas chromatograph-mass spectrometer. The method proposed showed good linearity over the concentration range tested and correlation coefficients ranged from 0.96 to 0.99 for all the analytes. The reproducibility and repeatability of the method was estimated between 1.29 and 4.02%. With no a pre-concentration step and with a much reduced analysis time, all the analyzed compounds showed detection and quantification limits that were lower than those observed with other methods found in the bibliography. Except for pentachlorophenol due to its poor absorptivity in polydimethysiloxane, in red wines, LOD ranged between 7.56 and 61.56 pg/l, and LOQ ranged between 17.21 and 205.11 pg/l; while in white wines, the LOD ranged between 5.82 and 30.50 pg/l and LOQ ranged between 19.41 and 101.61 pg/l. These concentrations were always lower than their respective olfactory thresholds values.

Kinetic analysis of a model of the sarcoplasmic reticulum Ca-ATPase, with variable stoichiometry, which enhances the amount and the rate of Ca transport.

The sarcoplasmic reticulum (SR) Ca-dependent adenosinetriphosphatase (Ca-ATPase) actively transports Ca2+ from the myoplasm to the SR lumen. Under optimal conditions a 2:1 stoichiometry of Ca transport/ATP hydrolysis has been observed, but lower stoichiometries have been reported under several circumstances. A lower stoichiometry under conditions of high Ca2+ load, although thermodynamically less efficient, could in theory increase the rate and the maximal amount of Ca uptake. We analysed, by computing simulation, the transient kinetics of a model of the SR Ca-ATPase with variable stoichiometry. The model is based on current experimental reports and includes the most relevant properties of the system. The results show an acceleration in the rate of Ca uptake, an increase in the net Ca transport, and an increase in the rate of [Ca2+] reduction in the medium, which might be physiologically useful to increase the rate of Ca pumping at high Ca load of the sarcoplasmic reticulum.

Effect of carticaine on the sarcoplasmic reticulum Ca2+-dependent adenosine triphosphatase.

The sarcoplasmic reticulum Ca2+-ATPase (calcium-dependent adenosine triphosphatase) transports Ca2+ from the myoplasm to the reticulum lumen at the expense of free energy from ATP hydrolysis. Carticaine is a local anesthetic of frequent use in dentistry which is now entering other clinical fields. We studied the action of carticaine on the sarcoplasmic reticulum (SR) skeletal muscle Ca2+-ATPase. SR vesicles from rabbit fast skeletal muscle were used. Carticaine inhibits the enzymatic activity. The inhibition of the enzymatic activity depends on pH, [Ca2+] and the presence of calcimycin. Half-maximal carticaine concentration that inhibits the ATPase activity tends to a maximal value upon increasing [Ca2+]. Carticaine concentrations required to inhibit the enzymatic activity at myoplasmic calcium concentration are lower than usual clinical doses: Ki=6.0+/-1.4 mM carticaine (n=5) for 0.1 microM [Ca2+]. ATP-dependent calcium uptake is also inhibited by the local anesthetic: Ki=30.5+/-3.4 mM (n=4). Besides, carticaine inhibits the phosphorylation of the enzyme by inorganic phosphate (Pi): Ki=20.0+/-3.4 (n=5) - 33.2+/-4.6 (n=4) mM, for [Pi] 1-4 mM. Carticaine increases the membrane permeability to Ca2+. Ca2+ efflux from preloaded vesicles is prevented by Ca2+ and Mg2+. Our results suggest that the diffusion of the local anesthetic into muscle fibers might trigger undesired effects such as sustained contraction of the masticatory muscles.

Method to determine the authenticity of aroma of saffron (Crocus sativus L.).

A thermal desorption-gas chromatography-mass spectrometry technique was applied to 252 different Spanish saffron samples (from La Mancha and Teruel). The average safranal content made up of 60% of the volatile fraction of the saffron. All the chromatograms obtained showed an interval between 8 and 18 min (retention time of safranal +/- 5 min) in which the silhouette of the chromatographic peaks was similar in all the samples. Therefore this interval can be used as a "fingerprint." Two examples are given which demonstrate the ability to use this "fingerprint" to detect adulteration.

Calcium additional to that bound to the transport sites is required for full activation of the sarcoplasmic reticulum Ca-ATPase from skeletal muscle.

The sarcoplasmic reticulum Ca-ATPase is fully activated when approximately 1 microM [Ca2+] saturates the two transport sites; higher [Ca] inhibits the ATPase by competition of Ca-ATP with Mg-ATP as substrates. Here we describe a novel effect of EGTA and other chelators, raising the possibility of an additional activating effect of Ca in the sub- or low microM range. Sarcoplasmic reticulum membranes were isolated from rabbit skeletal muscles. The ATPase activity was measured after incubation at 37 degreesC in 3 mM ATP, 3 mM MgCl2, 50 mM MOPS-Tris (pH 7.2), 100 mM KCl, and variable CaCl2, EGTA and calcimycin. In the absence of added EGTA and Ca the ATPase activity is high due to contaminant Ca. The determination of the ATPase activity in the presence of increasing amounts of EGTA, without added Ca, yields a decreasing sigmoidal function. Ki ranged between 20 and 100 microM, depending on the enzyme concentration. Pi production is linear with time for several [EGTA] yielding suboptimal ATPase activities, which are inhibited by thapsigargin. These suboptimal Ca-ATPase activities are inhibited by preincubation of the enzyme in EGTA, at pH 7.2. This effect increases upon increasing EGTA concentration and preincubation time. The inhibitory effect of the previous exposure of the enzyme to EGTA is partially but significantly reverted by increasing [Ca2+] during incubations. Calcimycin and EDTA have similar effects as EGTA when added in preincubations. The effect of calcimycin is fully reverted by optimal [Ca2+] in incubations. The effects of EGTA, EDTA and calcimycin in preincubation are not additive. The results suggest that an additional calcium, lost during preincubations from a site with affinity near 1 microM, is necessary for full activation of the ATPase.