Ultrahigh-Performance Liquid Chromatography (UHPLC)-Tandem Mass Spectrometry (MS/MS) Quantification of Nine Target Indoles in Sparkling Wines.

An ultrahigh-performance liquid chromatography (UHPLC)-tandem mass spectrometry (MS/MS) method was developed for the simultaneous determination of nine target indoles in sparkling wines. The proposed method requires minimal sample pretreatment, and its performance parameters (accuracy, repeatability, LOD, and matrix effect) indicate that it is suitable for routine analysis. Four indoles were found at detectable levels in commercial Cava samples: 5-methoxytryptophol (5MTL), tryptophan (TRP), tryptophan ethyl ester (TEE), and N-acetylserotonin (NSER). Two of them, NSER and 5MTL, are reported here for the first time in sparkling wines, with values of 0.3-2 and 0.29-29.2 µg/L, respectively. In the same samples, the contents of melatonin (MEL), serotonin (SER), 5-hydroxytryptophan (5-OHTRP), 5-hydroxyindole-3-acetic acid (5OHIA), and 5-methoxy-3-indoleacetic acid (5MIA) were all below the corresponding limits of detection.

Changes in RNA catabolites of sparkling wines during the biological aging.

In this study the catabolites derived from RNA degradation were assessed in Cava sparkling wines as a consequence of lees autolysis. For this purpose, the changes in the content of adenosine, guanosine, inosine, uridine, hypoxanthine, xanthine, and uric acid were determined by UHPLC-MS/MS, in sparkling wines produced on industrial scale, and aged for 4 years. Uridine is the main nucleoside, and its content increases whenever lees cells are present (sur lie aging). Purines seem to have a fermentative origin, with xanthine the most abundant one. When RNA catabolite amounts in sparkling wines aged with or without lees are compared over time, it can be concluded that lees and their cell degradation play an important role in the evolution of Cava; when lees are removed, RNA catabolite amounts remain unchanged.

Sparkling wines: features and trends from tradition.

Sparkling wines contain at least three CO2 pressure bars at 20°C. Carbonic gas is required to have an endogenous origin, obtained via a second fermentation, in the following European categories: sparkling wines and quality sparkling wines. In these types of sparkling wines, high gas pressure, together with other wine components, enables them to produce effervescence and foam when poured into the glass. The most commonly used grape varieties are Chardonnay and Pinot. Elaboration consists of two phases. In the first phase, the base wine is obtained after applying white vinification. The second phase consists of refermenting the wine, either in the bottle (champenoise or traditional method) or in isobaric tanks (Charmat method). The second fermentation requires the addition of "liqueur de tirage" to the base wine. The sparkling wines have a special biological aging or aging sur lies. As sparkling wines remain in contact with the lees, they develop sensory notes such as toasty, lactic, sweet, and yeasty, which can be attributed to proteolytic processes, components that would serve as the substrate for chemical and enzymatic reactions and to causes related with release-absorption between cell walls and the wine.

Ultrastructural changes of sparkling wine lees during long-term aging in real enological conditions.

Ultrastructural changes of lees of three series of sparkling wines produced using the traditional method during long-term aging (4 years) were assessed by high-pressure freezing in combination with transmission electron microscopy. The stratified structure of the cell wall disappeared throughout aging. After 18 months, the microfibrous material of the cell wall appeared more diffuse and the amorphous midzone of the inner wall layer was progressively degraded. From 30 months onward, the cell wall consisted of a tangled structure of fibers. In spite of these changes, the cell wall of yeasts remained unbroken at 48 months of wine aging. Cell membrane breakage was observed for the first time in lees of Saccharomyces cerevisiae. An increase in the thickness of the periplasmic space owing to plasmolysis and of the number of cells with less cytoplasmic content was observed during aging. Morphological evidence of microautophagy was detected for the first time in S. cerevisiae in enological conditions.

Characterisation of volatile composition of white salsify (Tragopogon porrifolius L.) by headspace solid-phase microextraction (HS-SPME) and simultaneous distillation-extraction (SDE) coupled to GC-MS.

Two analytical procedures were applied, HS-SPME and SDE, coupled to GC-MS to analyse the volatile composition of white salsify. More than 80 of volatile compounds which belonged to distinct chemical families were analysed. SDE led to the identification of mainly high molecular weight sesquiterpenes, acids and esters. Given that SDE involves high temperatures, heat-sensitive compounds may undergo chemical alteration or artefacts may appear. Results obtained show that SPME was useful for the analysis of alcohols and hydrocarbons of low molecular weight and high volatility that are involved in the characteristic volatile profile of salsify and its sensory perception.

Changes in the sorption of diverse volatiles by Saccharomyces cerevisiae lees during sparkling wine aging.

The volatile profile of sparkling wine is influenced by the retention and release of volatile compounds by lees during the aging process. Here we attempted to identify the volatiles that are most retained by lees in aging conditions and to study how their sorption varies during aging. We estimated the lees sorption capacity for several representative volatile compounds in sparkling wine samples at a range of time points during aging by assessing the volatiles sorbed on the lees surface and those present in the corresponding wines. The sorption of volatiles was proportional to their hydrophobicity, and their retention by the lees surface changed during aging. The sorption of less hydrophobic compounds decreased after the first 2 months of aging, while that of the most hydrophobic volatiles increased until 18 months, and decreased dramatically thereafter. These results indicate that the length of aging on lees determines the type and the amount of wine volatiles removed with lees in the disgorging step. While most polar aromas seem to be released from the lees surface at the earliest stages of aging, highly hydrophobic compounds and esters in general are progressively retained and subsequently desorbed into wine. Changes in the physicochemical properties of the lees cell surface were monitored during aging, but these could explain only the decrease in the sorption of less hydrophobic compounds.

Antioxidant activity of lees cell surface during sparkling wine sur lie aging.

Given the importance of the interactions between wine and lees cell surface during sparkling wine aging, and in view of recent results proving the antioxidant potential of yeast cell wall biomolecules, the antioxidant capacity of lees cell surface was investigated to establish its possible role in the antioxidative effect of lees. The surface antioxidant activity of lees from wines with different aging periods was determined on the whole cell by two widely used methods (DPPH and FRAP assays), obtaining maximum values of 24.5micromol Trolox/g cells (fresh weight) by the DPPH assay, and 21.3micromol Trolox/g cells (fresh weight) by the FRAP assay. Lees surface antioxidant activity was influenced by base wine characteristics and inversely related to sur lie aging period. Conversely, the percentage depletion of lees surface antioxidant activity during aging was mainly determined by the length of aging, regardless of wine characteristics. To examine the influence of cell wall thiol groups and adsorbed polyphenols on lees' protective effect, their presence on cell surfaces was assessed. They accounted for 25+/-11% and 54+/-7% of the antioxidant activity measured by DPPH, respectively, and 0.3+/-0.1% and 39+/-8% measured by FRAP, respectively. Only a part of the remnant antioxidant activity of lees surface measured by FRAP could be theoretically explained by the presence of cell wall mannans.

Evolution of sesquiterpene hydrocarbons in virgin olive oil during fruit ripening.

Despite the potential of sesquiterpene hydrocarbons in olive oil authentication, their metabolism in Olea europaea is poorly understood, and little is known about their biochemical regulation in olives as a function of ripening. To ascertain some metabolic relationships between sesquiterpene hydrocarbons and olive ripening, the content of sesquiterpene hydrocarbons was assessed in virgin olive oils from two olive varieties grown in the same geographical area and produced at different harvesting periods. During the ripening, the accumulation of sesquiterpenes in the olive itself, and thus in the oil, differed according to their molecular structure: bicyclic sesquiterpenes, showed decreasing concentrations the later the harvest, while acyclic farnesene-like compounds progressively increased through the olive ripening process. This is first evidence that the accumulation of sesquiterpene hydrocarbons in olive, and hence in olive oil, is modulated during ripening. Therefore, the degree of ripening of olives should be taken into consideration when considering the sesquiterpenic profile of virgin olive oil for their authentication.

Surface properties of Saccharomyces cerevisiae lees during sparkling wine ageing and their effect on flocculation.

Cell surface properties were determined for the first time for lees of Saccharomyces cerevisiae, and they proved to be significantly dependent on the time of sparkling wine ageing on lees. Cell surface hydrophobicity decreased from approximately 44% to 11% of affinity to hexadecane. Electron acceptor capacity (calculated as % affinity to ethyl acetate-% affinity to decane) increased from approximately -27% to 10 % and zeta potential from approximately -4 to 6mV. Mannoproteins are released from the external cell wall layer during the autolytic process, as confirmed by TOF-SIMS analysis of the cell wall chemical composition. This could explain the modifications of lees cell surface properties observed during ageing. Lees flocculation capacity, calculated by the Helm's method, decreased during the period of ageing from approximately 88 % to 40%. Stepwise multiple regression analysis indicated that lees surface hydrophobicity was the main determinant of lees flocculation capacity, explaining 70.3% of the variability (p<0.01), followed by far by the absolute values of zeta potential, accounting for 9.9% of the variability (p<0.05).

Assessment of volatile and sensory profiles between base and sparkling wines.

This paper focuses on the study of the volatile, olfactometric, and sensory composition of base wines and their corresponding sparkling wines (14-24 months aging) obtained at semi-industrial scale during three consecutive harvests. The sensory profile of sparkling wine is more complex than that of base wine, with toasty, lactic, sweet, and yeasty notes being described by the panelists and an even sharper increase of these tastes in the cava reserve. On the other hand, during the second fermentation and subsequent aging in contact with lees, some compounds such as acetate and ethyl esters decrease in amount while others such as norisoprenoids, acetal, diacetyl, and furans appear or increase over time. These volatile compounds could be responsible for the sensory profile depending on their notes, as determined by sniffing. The differences in volatile composition are responsible for the changes observed in the sensory profile of cava with respect to base wine.

Analysis of sparkling wine lees surface volatiles by optimized headspace solid-phase microextraction.

During sparkling wine aging, a narrow interaction is established between wine and lees of second fermentation, which remain in contact during long periods. In order to contribute to the knowledge on this interaction, volatile compounds retained by lees were described in this study. With this aim, solid phase microextraction (SPME) conditions were optimized in order to increase the sensibility for the analysis of volatiles sorbed by lees. This allowed proving the capacity of sparkling wine lees of second fermentation to retain several volatile compounds related with wine aroma: esters, aldehydes, norisoprenoids and terpenes known for their positive flavor impact were found in lees headspace. Most of them were previously described in "Cava" sparkling wine, while some compounds, such as the tentatively identified trimethyl tetrahydronaphthalenes, were not previously identified in yeasts or wine.

Influence of olives' storage conditions on the formation of volatile phenols and their role in off-odor formation in the oil.

Volatile phenols are spoilage compounds of many foods, which have also been detected in the volatile fraction of defective virgin olive oils (VOOs). However, their formation in olive fruits or VOOs, as well as the factors affecting their production, has yet to be elucidated. In the present study, the evolution of volatile phenols was monitored for the first time in VOOs obtained from olives stored during different periods under two different conditions of limited aerobiosis. Moreover, their odor activity values (OAVs) in VOO samples were calculated as a first assessment of their sensory importance, and the microbiological profile of the olives' surface was evaluated at each sampling point in order to clarify the possible causes of volatile phenols formation. Although volatile phenols seem to acquire their sensory significance at advanced stages of olives' alteration, they were significantly correlated to the time of olives' storage and in accordance with sensory evaluation, indicating that they could be considered as analytical indices of olive fruits' degradation during storage, likely reflecting the microbiological activity.

Determination of volatile phenols in virgin olive oils and their sensory significance.

Volatile phenols are strong odorants produced by microbial activity and reported in several foods, but very scarce information is available on their presence in virgin olive oils (VOOs) and on their relation with VOO chemical and sensory quality. In the present paper, a factorial experimental design was applied for the development of a suitable solid-phase microextraction-gas chromatography/mass spectrometry (SPME-GC/MS) analytical method for the analysis of volatile phenols in olive oil. The memory effects demonstrated by SPME fibres required the optimization of desorption conditions to minimize experimental errors. A series of nine volatile phenols were identified and quantified for the first time in VOOs by analyzing samples with distinct off-flavours. Their limits of detection and quantification (microg/kg) were largely below the odour detection thresholds (ODTs) calculated in this study (mg/kg), confirming the capacity of the technique to assess the target compounds at early stages of the oil sensory alteration. The odour activity values (OAVs) of volatile phenols were calculated in VOOs facilitating a first assessment of their potential importance in the aroma of the product.

Assessment of some diterpenoids in commercial distilled gin.

In the present study the qualitative and quantitative determination of diterpenoids in commercial distilled gin was carried out. This widely consumed juniper-based spirit is aromatized using Juniper (Juniperus communis) berries. Although juniper reportedly contains several diterpenic compounds, no studies have addressed the diterpenic composition of juniper-based spirits or beverages. With this objective, here we followed a multilevel factorial experimental design to optimize a direct immersion-solid phase microextraction (DI-SPME) method coupled to gas chromatography/mass spectrometry and analyzed eight commercial brands of gin. With total concentrations ranging from 10 to 190 microg L(-1), manool, manoyl oxide and trans-totarol were the most abundant diterpenoids of the 10 identified or tentatively identified at variable but not negligible concentrations in the distilled gin samples. The diterpenic composition allowed the brands to be differentiated. This indicates that these compounds contribute to the sensory characteristics of the distinct commercial brands, thus guaranteeing the authenticity and consequently the quality of the product.

Different commercial yeast strains affecting the volatile and sensory profile of cava base wine.

36 semi-industrial fermentations were carried out with 6 different yeast strains in order to assess differences in the wines' chemical and volatile profile. Two of the tested strains (Y3 and Y6) showed the fastest fermentation rates throughout 3 harvests and on 2 grape varieties. The wines fermented by three of the tested strains (Y5, Y3 and Y4) stand out for their high amounts of esters and possessed the highest fruity character. Wines from strains producing low amounts of esters and high concentrations of medium chain fatty acids, higher alcohols and six-carbon alcohols were the least appreciated at the sensory analysis. The data obtained in the present study show how the yeast strain quantitatively affects the final chemical and volatile composition of cava base wines and have repercussions on their sensory profile, independently of must variety and harvest year.

Monoterpene and sesquiterpene hydrocarbons of virgin olive oil by headspace solid-phase microextraction coupled to gas chromatography/mass spectrometry.

In the present article, a headspace solid-phase microextraction method coupled to GC/MS was developed and applied for the simultaneous determination of mono- and sesquiterpenic hydrocarbons in virgin olive oils of different olive variety and geographical origin. Analysis of various oils resulted in the simultaneous detection of 15 monoterpenes and 30 sesquiterpenes. Some of these hydrocarbons were previously reported to be constituents of virgin olive oil terpenoid fraction, although we also detected some terpenic hydrocarbons that have not previously been documented as present in virgin olive oil. Significant differences were detected in the proportion of terpenic compounds in oils obtained from different olive varieties grown in different geographical areas. The monoterpene, and particularly the sesquiterpene composition of olive oil may be used to distinguish samples from different cultivar and geographical areas.

Characterization of volatiles in different dry gins.

A HS-SPME method coupled to GC-MS was developed and applied for the qualitative and semiquantitative characterization of distilled gin volatile fraction. Sampling, chromatographic conditions, and method performances were evaluated, and the developed method was applied as a comparative study of some of the most popular commercial London Dry Gins and other gins with geographic denominations. During this study, 70 components of the gins' volatile fraction were isolated, tentatively identified or identified by reference compounds. They were mainly represented by mono- and sesquiterpenic compounds, which were quantitatively determined. The comparative study of London Dry Gins and gins with geographical indication permitted clear differentiation between the gins with geographical indication and one of the London Dry Gins tested.

Simultaneous determination of volatile and semi-volatile aromatic hydrocarbons in virgin olive oil by headspace solid-phase microextraction coupled to gas chromatography/mass spectrometry.

A reliable, simple and relatively fast method for the simultaneous determination of volatile and semi-volatile aromatic hydrocarbons in virgin olive oil was developed, based on headspace solid-phase microextraction (HS-SPME). The investigation regarded eco-contaminants such as alkylated monoaromatic hydrocarbons from C1- to C4-benzenes and light polyaromatic hydrocarbons up to four aromatic rings. Sampling and chromatographic conditions were optimized by using standard solutions in deodorized olive oil and the analytical performances of the method were determined. The proposed method was then applied to real samples of virgin olive oil were the target hydrocarbons could be identified and quantified. Several of them had not been previously quantified in virgin olive oil. Moreover, by the analysis of olive oil samples an additional number of C4-benzenes could be tentatively identified.

Assessment of the volatile composition of juices of apricot, peach, and pear according to two pectolytic treatments.

The behavior of volatile compounds according to two enzymatic treatments applied during the manufacture of fruit juice is described. More than 80 compounds were detected of a wide range of chemical families (alcohols, aldehydes, ketones, terpenoids, esters, norisoprenoids, ...). Theaspirane and alpha-isophoron were tentatively identified for the first time in apricot and peach fruits. The enzymes used, for extraction or clarification of fruit juices, modified the polysaccharides separated by molecular weight and the content of soluble polysaccharides. This could indicate differences in the fruit juice matrix, which could be related to observed changes in the volatile profile. In apricot, the enzymes enhanced the juice in terpenes and norisoprenoids as varietal compounds. In peach and pear, the enzymes used did not favor the amount of lactones and decadienoate esters, the character impact compounds, respectively.

Flavonoid metabolites and susceptibility of rat lipoproteins to oxidation.

Flavonoids are ingested with vegetables and beverages and exert a beneficial effect on cardiovascular disease. Studies in animals in vitro and in humans ex vivo on the resistance of lipoproteins to oxidation are not consistent and the mechanisms by which flavonoids protect against atherosclerosis are a matter of debate. In the present study, we investigated the effects of administering diets containing 0.3% (wt/wt) quercetin, 0.3% (wt/wt) catechin, or 35% (vol/wt) dealcoholated red wine (DRW) for 10 days in healthy rats on markers of oxidative damage in lipoproteins and in plasma. The antioxidant levels in low-density lipoproteins (LDL) or the lag phase, oxidation rate, and maximum level of conjugated dienes during ex vivo LDL oxidation did not differ between control and treated rats. Plasma levels of alpha-tocopherol and retinol were similar in all groups. The total antioxidant status of the plasma from rats fed either quercetin or DRW diet was higher than in control rats. Only glucuronide and sulfate compounds of quercetin were detected in plasma from rats fed the quercetin-rich diet, and no flavonoids or their metabolites were detected in plasma or LDL from rats fed the catechin- or the DRW-rich diet. No significant differences in malondialdehyde or in conjugated dienes in plasma were observed. These results indicate that although metabolites from quercetin are present in plasma, they are not detected in lipoproteins and do not modify the level of other antioxidants. In conclusion, in the absence of any pathology or of oxidative stress the intake of quercetin, catechin, or DRW did not protect lipoproteins from oxidation ex vivo.