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Analytica Chimica Acta (v.563, #1-2)

Contents (pp. vii-ix).

Calendar of forthcoming meetings (pp. n1).

Filler Ad.: ACAEES (pp. ii).

Outside front cover - Editorial Board (pp. co1).

Foreword by Véronique Cheynier; Christian Ducauze; Douglas N. Rutledge (pp. 1-1).

Chromatic and structural features of blue anthocyanin-derived pigments present in Port wine by Joana Oliveira; Celestino Santos-Buelga; Artur M.S. Silva; Victor de Freitas; Nuno Mateus (pp. 2-9).

A new group of blue anthocyanin-derived pigments was recently found to occur in ageing Port wines and were named as portisins. The UV–vis spectrum of these pigments was found to have a λ_max around 570nm that is bathochromically shifted from that of original anthocyanins, exhibiting a more bluish hue in acidic solution. Three portisins were synthesised through the reaction between malvidin 3- O-glucoside, malvidin 3- O-coumaroylglucoside and cyanidin 3 -O-glucoside-pyruvic acid adducts and catechin in presence of acetaldehyde. These portisins were structurally and chromatically characterized (pH, SO₂ stability assays and determination of the molar extinction coefficient) and showed to have high resistance to the attack of water or bisulfite, specially when compared to malvidin-3-glucoside. This portisin resistance to discoloration may be explained by a higher protection of the chromophore group against the water or bisulfite nucleophilic attack that gives rise to the hemiacetal form, which is usually colourless. Portisins also showed to have a very high colorant capacity as they displayed a higher molar extinction coefficient than anthocyanins and the respective pyruvic acid adducts. These chromatic characteristics may help better understand the contribution of these anthocyanin-derived pigments to the overall wine colour.

Keywords: Portisin; Blue; Pigment; Colour; Red wine

Influence of structure on the ionisation constants of anthocyanin and anthocyanin-like wine pigments by Robert E. Asenstorfer; David F. Lee; Graham P. Jones (pp. 10-14).

The macro ionisation constants of malvidin-3-glucose, vitisin A and the two pairs of diastereoisomers of 8,8-ethyl-linked pigments formed between malvidin-3-glucose and catechin or epicatechin were determined using high voltage paper electrophoresis (HVPE) and compared as a function of their chemical structures. Their UV–vis spectral characteristics were also compared. The low p K_a1 value of vitisin A (0.95±0.10) compared to malvidin-3-glucose (1.76±0.07) reflects the extended delocalisation of the former due to the presence of the fourth ring and the electron withdrawing effect of the carboxyl group. The presence of the ethyl bridge at the 8-position of the 8,8-ethyl-linked malvidin-3-glucose–(epi)catechin give rise to slightly higher p K_a1 values (2.33–2.44) due to the electron donating effect of the alkyl group into the delocalised electron system. Similarly, the loss of a second proton, reflected by the p K_a2 values is influenced by the 8-ethyl substituent. Small differences in p K_a1 and p K_a2 values between the different diasterioisomers reflects the impact of variation in three-dimensional structures. p K_a1 and p K_a2 values for these compounds determined by HPVE agree well with those determined by UV–vis spectroscopy indicating that the latter are true ionisation constants rather than a composite of ionisation and hydration constants.

Keywords: Anthocyanins; Ionisation constants; High voltage paper electrophoresis; Wine pigments

Formation of anthocyanin–flavanol adducts in model solutions by M. Dueñas; H. Fulcrand; V. Cheynier (pp. 15-25).

The mechanism of direct reactions between malvidin 3-O-glucoside (Mv3glc) and a flavanol monomer, (−)-epicatechin, was studied in model solutions in the pH range 2.0–6.0. The direct reactions are strongly related to pH. The rate of Mv3glc and epicatechin disappearance increased with pH. Nucleophilic addition of epicatechin onto the flavylium form of Mv3glc took place at all pH values. At pH 2.0, the resulting intermediate flavene yielded a colourless Mv3glc-epicatechin adduct with an A-type linkage. In the range pH 3.2–6.0, two new products with UV–vis spectra characteristic of those of xanthylium ions and mass signals at m/ z 633 and 453, respectively, in the positive ion mode were detected. These products are postulated to be degradation products of the flavylium form of Mv3glc-epicatechin arising from the intermediate flavene. Moreover, above pH 4.0, oxidation of epicatechin promoted the formation of new pigments detected at m/ z 781, in the positive ion mode. These ions were attributed to epicatechin-Mv3glc adducts formed by addition of Mv3glc onto the epicatechin o-quinone as the loss of a 138amu fragment indicated a biphenyl or biphenyl-ether linkage between the flavanol B-ring and the anthocyanin A-ring.

Keywords: Anthocyanins; Malvidin 3-O-glucoside; Flavanol monomer; Xanthylium; Pigments; Biphenyl linkage

A first approach towards the relationship between grape skin cell-wall composition and anthocyanin extractability by A. Ortega-Regules; I. Romero-Cascales; J.M. Ros-García; J.M. López-Roca; E. Gómez-Plaza (pp. 26-32).

The composition of skin cell-walls from four different grape varieties ( Vitis vinifera L., cv. Cabernet Sauvignon, Merlot, Syrah and Monastrell, the later harvested in three different locations) has been studied, trying to correlate those differences found in cell-wall composition among varieties with their degree of ripeness and with the easiness of anthocyanin extractability. The results of an anthocyanin extractability assay showed that the anthocyanins from Monastrell grape skins might be more difficult to extract than those from the other varieties. The study of the cell-wall composition showed some differences in uronic acids, cellulosic glucose, proteins and polyphenol content of cell-walls among varieties, and also differences between the Monastrell grapes cultivated in different locations were detected. Little differences could be found among the different samples regarding the non-cellulosic neutral sugars. In trying to look if differences in the extractability index were related to cell-wall composition and to help to clarify some of the mechanism involved in the anthocyanin extractability, a multiple regression analysis was conducted. The results showed that a model could be built up explaining a high percentage of the variability in the extractability index, using the content of the different components of the skin cell-wall as independent variables. Following this approach, the differences on the easiness of anthocyanin extractability in grape samples could be based in differences in pectin and cellulose content, but also, differences occurring on arabinoxylan, arabinogalactan and xyloglucan could be of importance.

Keywords: Wine; Anthocyanin extractability; Cell-wall composition

Size-exclusion chromatography of procyanidins: Comparison between apple and grape procyanidins and application to the characterization of fractions of high degrees of polymerization by C. Le Bourvellec; M. Picot; C.M.G.C. Renard (pp. 33-43).

Apple procyanidins were fractionated on Toyopearl TSK-gel HW-40S or HW-50S resins eluted by an acetone:6M urea:acetic acid mixture. Determination of number average degrees of polymerization of the procyanidins in eluted fractions confirmed that in those conditions true molecular weight profiles were obtained. This method was applied to polyphenols extracted from cider apples of different varieties at various stages of maturity, from pear juice and from grape seeds. They confirmed existence of very highly polymerized fractions in perry pears and in certain apple varieties. During post-harvest maturation of apples, the number average degree of polymerization of their procyanidins decreased slightly, and their size-exclusion chromatograms showed disappearance of fractions at low K_av values.

Keywords: Tannins; Molecular weight; Maturation; Malus domestica; Borkh

Recovery of catechins and proanthocyanidins from winery by-products using subcritical water extraction by Matilde García-Marino; Julián C. Rivas-Gonzalo; Elena Ibáñez; Concepción García-Moreno (pp. 44-50).

The aim of this work was to study the recovery of catechins and proanthocyanidins from grape seeds obtained as by-products from wineries using superheated water. The effect of temperature in the extraction process was studied. For this purpose, five different assays were carried out. In three of them, samples were extracted once with water at 50, 100 and 150°C, respectively, working at pressures around 1500psi to keep the water in the liquid state. In the fourth, samples were treated twice using sequentially 50 and 100°C, and in the fifth a three-stage sequential extraction was used at 50, 100 and 150°C. The composition of the extracts obtained was performed by HPLC–DAD–MS. The results were compared with those obtained using conventional analytical extraction with methanol/water (75:25) at atmospheric pressure. The results showed that superheated water is a good solvent for extracting flavanols, in some cases better than methanol/water (75:25). In general, major recoveries were found when the material was submitted to three sequential extractions at 50, 100 and 150°C, but selective extractions of compounds with different degrees of polymerisation can be achieved using one-step extraction at different temperatures. Better recoveries for flavanol dimers and trimers, showing higher antioxidant activity, were obtained using a single extraction at 150°C. Furthermore, gallic acid, with antioxidant characteristics similar to the catechin and epicatechin monomers, is obtained in greater quantities by a single extraction at 150°C. The greater the temperature the greater extraction of gallic acid, which reaches approximately 70% of the total of polyphenols extracted.

Keywords: Subcritical water extraction; Catechins; Proanthocyanidins; Winery by-products; Grape seed; Polyphenols

Validation of an analytical method for the solid phase extraction, in cartridge derivatization and subsequent gas chromatographic–ion trap tandem mass spectrometric determination of 1-octen-3-one in wines at ngL⁻¹ level by Laura Culleré; Juan Cacho; Vicente Ferreira (pp. 51-57).

An analytical method for the quantification in wines of the strong-smelling compound, 1-octen-3-one, has been developed. 1-Octen-3-one is extracted by passing 90mL of wine through a solid phase extraction (SPE) cartridge packed with 90mg of LiChrolut-EN resins. Wine major volatiles and potential interferences are removed by washing up with 9mL of a 40% methanol–water solution containing 1% NaHCO₃. The ketone is subsequently derivatised with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine (PFBHA) to form its two isomeric oximes. Reaction takes place in the same cartridge at room temperature (25°C) in 15min. The excess of reagent is then removed by washing with 20mL of a 0.05M sulphuric acid solution. The corresponding oximes are further eluted with pentane and determined by gas chromatography coupled with ion trap tandem mass spectrometry (GC–MS/MS) by non-resonant fragmentation of the m/ z 140 parent ion and subsequent analysis of the corresponding daughter ions ( m/ z 77, 79, 94 for the trans and m/ z 77 and 79 for the cis-isomer, respectively). The analytical signal is highly specific and in all samples the obtained MS spectrum are fully coincident with those from synthetic standards. Recoveries were in all the studied cases 100%. Precision (measured as R.S.D.) is better than 5%, linearity holds at least up to 900ngL⁻¹ ( r²=0.9990), and the method detection limit is 0.75ngL⁻¹, well below the olfactory threshold of the compound (15ngL⁻¹). The range of concentrations found in normal, non-spoiled dry wines goes from 7 to 61ngL⁻¹. Sensory experiments have shown, however, that such differences do not seem to be enough to explain sensory differences.

Keywords: Aroma; 1-Octen-3-one; Wine; Solid phase extraction; Gas chromatography–mass spectrometry–mass spectrometry; Derivatization

Evidence for an alternative biogenetic pathway leading to 3-mercaptohexanol and 4-mercapto-4-methylpentan-2-one in wines by R. Schneider; F. Charrier; A. Razungles; R. Baumes (pp. 58-64).

The present investigation ascertains a new pathway leading to 3-mercaptohexanol (3Mhol) and 4-mercapto-4-methylpentan-2-one, starting from conjugated carbonyl compounds, alternative to the biogenetic route already demonstrated in wines from cysteinylated and gluthationylated precursors present in grapes, [D₈]- E-2-hexenal and [D₁₀]-mesityl oxide, possible precursors of 3-mercaptohexanol and 4-mercapto-4 methylpentan-2-one by sulphur addition, were synthesized and added to a Melon B. must, prior to its fermentation at laboratory scale. Volatile thiols extracted from the wine were analyzed by GC–MS. Peaks corresponding to [D₈]-3Mhol and [D₁₀]-4MMP were detected, showing their origin from [D₈]-2-hexenal and [D₁₀]-mesityl oxide added to the must. Furthermore, an accurate quantification of the proportion between [D₈]-3-Mhol and natural 3Mhol showed that this pathway was minor in our fermentation conditions (about 10% of total 3Mhol).

Keywords: Carbonyl comounds; Fermentation; Biogenetic pathway; Wine; 3-Mercaptohexanol; 4-Mercapto-4-methylpentan-2-one

Flavan-3-ol compositional changes in grape berries ( Vitis vinifera L. cv Cabernet Franc) before veraison, using two complementary analytical approaches, HPLC reversed phase and histochemistry by Y. Cadot; M.T. Miñana Castelló; M. Chevalier (pp. 65-75).

Several studies have shown that the proanthocyanidins are responsible for some major flavour properties of the red wines such as colour, bitterness and astringency. Nevertheless, their synthesis and properties are still misunderstood. Some studies have suggested that the proanthocyanidin content is set before veraison.This paper describes the setting of the proanthocyanidins, until the veraison stage, and the relations that exist with the environment. The flavan-3-ol composition was monitored from the end of the berry set until mid veraison, on two plots known to induce a different behaviour of the vine. The study considered the value of the parameters for a homogeneous stage of development. The composition in proanthocyanidins in skin was determined by HPLC reversed phase, after fractionation and thiolysis. A further information about the proanthocyanidins was obtained by histochemistry. The sections, 2μm thick, were stained according to the 4-dimethylaminocinnamaldehyde method.The interest and the limits of these approaches are described and discussed. Moreover, it is shown that the linking of these two analytical techniques which associates quantitative data and the spatializing of the tannins in cellular tissue could improve the understanding of the setting of the proanthocyanidins content. Finally, this original approach pointed out the importance of the period previous ripening in the synthesis of flavan-3-ols in the skin; the heterogeneity among berries was in relation with the stage of development but some other factors, as light exposition could also play a role.

Keywords: Vitis vinifera; Grapevine; Flavan-3-ols; Proanthocyanidins; Histochemistry; 4-Dimethylaminocinnamaldehyde

Condensation products derived from flavan-3-ol oak wood aldehydes reaction by Marie-Françoise Nonier; Isabelle Pianet; Michel Laguerre; Nicolas Vivas; Nathalie Vivas de Gaulejac (pp. 76-83).

In this first work, we report on a complete structural study of the condensation dimers formed in a wine like model solution (pH 3.5; H₂O:EtOH, 88:12; 14 days of reaction in the dark) between a catechin and a representative oak wood aldehyde:furfuraldehyde. Of the four dimers expected, three were formed in sufficient quantity to be isolated by liquid chromatography. In this first study, the regio- and stereo-chemistry as well as the 3D structure of these compounds were obtained by using NMR and molecular modelling techniques.

Keywords: Catechin; Furfuraldehyde; 3D structure; NMR; Molecular modelling

Analysis of phenolic compounds in Muscatel wines produced in Portugal by M.N. Bravo; S. Silva; A.V. Coelho; L. Vilas Boas; M.R. Bronze (pp. 84-92).

A liquid chromatography method associated with mass spectrometry and diode array, fluorescence and electrochemical detectors was used in order to study phenolic composition of Muscatel sweet wines from Setúbal region in Portugal. Samples were collected during winemaking production at different representative producers of this region. Total phenolic contents of samples were also determined using the Folin–Ciocalteu method. Mass spectrometry results show that atmospheric pressure chemical ionisation (APCI) in negative mode presents higher sensitivity for the majority of the compounds studied. Some phenolic acids, stilbenes as resveratrol and piceid, and flavonols as quercetin and quercetin glycosides were identified in these Muscatel wines. For resveratrol, piceid, gallic acid, protocatechuic acid, catechin and quercetin, fluorescence and electrochemical properties were used as complementary or alternative methods of detection. Differences in phenolic composition and total phenolic contents were found among samples collected.

Keywords: Muscatel wine; Phenolic compounds; Fluorescence detector; Electrochemical detector; Mass spectrometry; Folin–Ciocalteu

Phenolic composition and quality of white d.o.c. wines from Marche (Italy) by Emanuele Boselli; Massimo Minardi; Andrea Giomo; Natale G. Frega (pp. 93-100).

Two white d.o.c. wines produced with grape varieties typical from the region of Marche (Italy) were subjected to high performance liquid chromatography coupled with electrospray tandem mass spectrometry in order to characterize the phenolic fraction. Unlike Verdicchio, Passerina showed relatively high concentrations of tyrosol (up to 45mgL⁻¹) and quercetin, with the glucuronide (20mgL⁻¹), being more concentrated with respect to the free form (6mgL⁻¹). Conversely, Verdicchio showed high concentration of 3,4-dihydroxyphenylethanol (hydroxytyrosol) and hydroxycinnamic acids, such as caffeic, caftaric, coumaric and 2-S-glutathionylcaftaric acid and remarkable amounts of their esters with ethanol (total amount in the order of 30mgL⁻¹). The different composition of the phenolic fraction had a strong impact on the sensory properties, as confirmed by the sensory data elaborated with principal component analysis (PCA): the taste of burning and astringent was associated with caftaric, ethyl coumarate and 2-S-glutathionylcaftaric acid (thus with Verdicchio), whereas the taste of honey, fruity and exotic fruits was related to Passerina. The identification of 2-S-glutathionylcaftaric acid was performed with HPLC coupled with tandem mass spectrometry in the positive and negative electrospray ionization mode and is discussed here.

Keywords: White wines, Verdicchio; Passerina; Phenolics; High performance liquid chromatography; Mass spectrometry; Sensory analysis; Principal component analysis

Determination of the phenolic composition of sherry and table white wines by liquid chromatography and their relation with antioxidant activity by M.S. Fernández-Pachón; D. Villaño; A.M. Troncoso; M.C. García-Parrilla (pp. 101-108).

The purpose of this work is to study possible relationships between the antioxidant activity (AA) of 13 table white and 9 sherry wines and their phenolic composition. Twenty phenolic compounds were determined by liquid chromathography (LC). AA has been determined by different methods, including 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH) and Oxygen Radical Absorbance Capacity (ORAC). The AA of pure phenolic standard compounds has also been measured. Statistical study has been performed using multiple regression analysis and artificial neural networks to ascertain if a specific phenolic composition accounts for a higher AA. Certain phenolic compounds exhibit a high linear correlation with the overall AA of wines. For example, gallic acid, glucoside of cutaric acid and tyrosol as determined by the ORAC method. If we study AA values obtained by ABTS and DPPH methods, a good correlation is obtained with (−)-epigallocatechin gallate and ethyl caffeate. The contribution of each phenolic compound to the AA was also examined. Gallic acid, protocatechuic acid, caffeic acid and tyrosol are the main contributors to the ORAC value. If we use ABTS and DPPH methods, the main contributors are gallic, caffeic and caftaric acids and (−)-epigallocatechin gallate.An equation calculated by Multiple Regression Analysis correlates ( r=0.9028) ORAC values of wines with concentration of certain phenolic compounds (gallic acid, protocatechuic acid, tyrosol, caffeic acid, (−)-epigallocatechin gallate, coumaric acid and 2-furaldehyde).

Keywords: Antioxidant activity; Wine; Phenolic compounds; Oxygen Radical Absorbance Capacity; 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid); 1,1-Diphenyl-2-picrylhydrazyl

Impact of prefermentative maceration on the phenolic and volatile compounds in Monastrell red wines by I. �?lvarez; J.L. Aleixandre; M.J. García; V. Lizama (pp. 109-115).

The effects of two prefermentation treatments (cold soak at 6–8°C and cold soak at 0–2°C with dry ice) and two different maceration times (4 and 8 days) in must and wine composition have been studied. Grapes from Vitis vinifera var. Monastrell were harvested at two maturation levels. Total phenolic, colour density, anthocyanins and fractioned anthocyanins, ionized anthocyanins, polymeric pigments colour, condensed tannins and aromatic compounds were estimated. The effects of temperature in the grape juice composition were determined with constant monitoring during the prefermentation step. Significant differences were found among the measured parameters in the grape juice and wine. Prefermentative maceration produces wines with higher phenolics compounds, anthocyanins, especially malvidin-3-glucoside, ionized and polymeric anthocyanins and aromatic compounds values compared to control wines. This increase is more significant when the prefermentation treatments are done with the use of dry ice and the must is produced from less mature grapes. The maceration time showed not to be significant during the process.

Keywords: Cryomaceration; Dry ice; Red wine; Phenolic compounds; Volatile composition

Phenolic compounds in red wine subjected to industrial malolactic fermentation and ageing on lees by Teresa Hernández; Isabel Estrella; Davinia Carlavilla; Pedro J. Martín-�?lvarez; M. Victoria Moreno-Arribas (pp. 116-125).

A liquid chromatography method with photodiode array and mass spectrometry detection was applied to study the changes in non-anthocyanin phenolic compounds from 47 red wines as a result of different technological processes. The procedures used include: malolactic fermentation in barrels or in stainless steel containers, the ageing of wines or not in the presence of lees, including periodic stirring of the lees, racking, clarification, cold stabilization and filtration. Wine samples were taken before and after malolactic fermentation and during the 3 months of the experiment until they had been aged for 14 months in oak barrels. Cluster analysis, analysis of variance and stepwise discriminant analysis were applied to the wine data. During malolactic fermentation, disappearance of the tartaric esters of caffeic acid (caftaric acid) and p-coumaric acid (coutaric acid) was detected, coinciding with an increase in the corresponding free acids. With the statistical treatments used, the greatest differences in the low molecular weight phenolic compounds in the wines were due to ageing time, whereas the winemaking practice did not significantly influence phenolic compounds. A significant change in the concentration of most phenolic compounds was observed in the wine at the end of the 14 months of ageing.

Keywords: Wine; Non-anthocyanin phenolic compounds; Liquid chromatography–Photodiode array detection; Liquid chromatography–Mass spectrometry; Malolactic fermentation; Ageing on lees

Relationship between polyphenolic composition and some sensory properties in red wines using multiway analyses by S. Preys; G. Mazerolles; P. Courcoux; A. Samson; U. Fischer; M. Hanafi; D. Bertrand; V. Cheynier (pp. 126-136).

Polyphenolic compounds are responsible for important sensory properties of red wines (especially colour, astringency and, to a lesser extent, bitterness). Various model solution studies have investigated the relationships between specific phenolic compounds and sensory perception. The purpose of the present study was to relate polyphenolic composition to sensory data for a very large number of different commercial wines, using multiway analyses. Two homogeneous populations of commercial red wines (61 French and 60 German wines) were analyzed. Thirty simple polyphenolic compounds (anthocyanins, flavonols and phenolic acids) and some red pigment derivatives have been quantified using direct injection by liquid chromatography (LC) coupled to diode array detection and mass spectrometry. Condensed tannins were analyzed using LC after thiolysis. Sensory perception was assessed using descriptive analysis by four trained panels (2 countries×2 years). We first checked the consistency of the sensory data both through the 2 assessment years, and through the two countries. Although reproducibility was high, especially through countries, slight scale factor differences and scale shifts were detected. A chemical consensus was then built using common components and specific weights analysis for both wine subsets, where the sensory variables of each panel were projected separately. Although data structure was very different between the two wine subsets, common main chemical-sensory relationships were confirmed. The hypothesis of a relationship between flavonol aglycones and bitterness was raised. A partial least squares regression was performed and predicted linearly astringency with a R² value of 0.80, but not colour intensity and hue, due to non-linear relationship and saturation of visual perception.

Keywords: Polyphenols; Wine; Sensory descriptive analysis (DA); Liquid chromatography (LC); Thiolysis; Multiway analyses

¹³C NMR analysis of polyphenol biosynthesis in grape cells: Impact of various inducing factors by Cassandrine Saigne-Soulard; Tristan Richard; Jean-Michel Mérillon; Jean-Pierre Monti (pp. 137-144).

¹³C NMR spectroscopy was used as a complement to high performance liquid chromatography (HPLC) or spectrophotometry to analyse stilbene and anthocyanin metabolism in grape cell cultures. Stilbene and anthocyanin biosynthesis are closely related. Anthocyanins are major components of grape quality whereas stilbenes play a crucial role in defence mechanisms and are strongly correlated to natural grapevine resistance. Stimulation of stilbene production by natural inducers offers an interesting alternative to the use of pesticides in plant protection strategies. NMR allowed us to analyse both stilbene and anthocyanin pathways by following step by step the incorporation of the labelled precursor [1-¹³C]l-phenylalanine along the entire biosynthetic routes. However, several intermediates of the anthocyanin biosynthetic pathways remained unobservable, providing evidence for metabolic channelling. The effect of various elicitors on stilbene and anthocyanin biosynthesis was also investigated. In complement to quantification of the end-products by HPLC and spectrophotometry,¹³C NMR studies provided information at the biosynthetic level. Sucrose addition stimulated the biosynthesis of anthocyanins without influencing stilbenes. Methyljasmonate and fungal elicitor strongly increased stilbene production through the activation of enzymes from phenylalanine ammonia-lyase to stilbene synthase. However, methyljasmonate showed an inhibitory effect on anthocyanin biosynthesis, suggesting the existence of a competition between the two pathways.¹³C NMR spectroscopy, combined with analytical techniques such as HPLC and spectrophotometry, provides a very interesting tool to better understand the mechanisms underlying the production of secondary metabolites and the metabolic processes of plant resistance induction by elicitation.

Keywords: ¹³; C NMR spectroscopy; Vitis vinifera; L.; Cell culture; Polyphenol biosynthesis; Plant resistance; Inducing factors

Characterization of the volatile fraction of young wines from the Denomination of Origin “Vinos de Madrid�? (Spain) by Mar Gil; Juan Mariano Cabellos; Teresa Arroyo; Marin Prodanov (pp. 145-153).

Twenty-five volatile compounds of 71 white, 53 rose and 70 red commercial young wines, representative for the young wines commercialized under the Denomination of Origin (DO) “Vinos de Madrid�? (Spain) were analyzed by gas chromatography-flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Odor activity values (OAV) of 21 of them were calculated, also. Twenty-four of the 25 determined odor compounds were found in the three types of wines, which means that the differences between white, rose and red wines are essentially quantitative, rather than qualitative. 4-Vinyl-guayacol was typical only for the white wines. Thirteen of the 21 odor compounds with determined OAVs had significant relevance to the global aroma of the white and rose and 12 of the red wines. Among them (in diminishing order of odor importance) ethyl octanoate, ethyl hexanoate, isoamyl acetate and isovaleric acid were the most determinant for the aroma of the white and rose wines, whereas, ethyl octanoate, isovaleric acid, isoamyl acetate and ethyl hexanoate were for the red wines. Discriminant statistic analysis showed that the isoamylic alcohols, higher major alcohols, ethyl hexanoate, acetates, ethyl octanoate, 4-vinyl-guaiacol and decanoic acid were the most significant compounds in the differentiation between the white, rose and red young wines. The same analysis showed correct classification of 96.6% of the wines (92.1% cross-validation), according to their type (white, rose or red).

Keywords: Young wines; Volatiles; Aroma; Gas chromatography; Denomination of Origin “Vinos de Madrid�?

Analytical characterization of the aroma of Tinta Negra Mole red wine: Identification of the main odorants compounds by R. Perestrelo; A. Fernandes; F.F. Albuquerque; J.C. Marques; J.S. Câmara (pp. 154-164).

A method for the simultaneous determination of major and minor volatiles composition in different types (dry, medium dry, sweet and medium sweet) of a young Tinta Negra Mole (TNM) monovarietal red wine from 2003 harvest has been validated. Wine samples preparation includes a dichloromethane liquid–liquid extraction followed by concentration under a nitrogen atmosphere. The extracted fraction was analysed by gas chromatography–mass spectrometry and give quantitative information for more than 86 analytes whose concentration range from few μgl⁻¹ to 259.1mgl⁻¹. The method enables high recovery of volatile compounds in wine good linearity with ( r²) values higher than 0.980 and good sensitivity. The limits of detection range from 0.003 to 0.534mgl⁻¹ and limits of quantification from 0.009 to 1.170mgl⁻¹.The method allows satisfactory determination of more than 80 compounds in the TNM red wines. These wines are characterized by a high content of higher alcohols, ethyl esters, fatty acids and lactones. The levels of sulphur compounds in Tinta Negra Mole medium sweet wines are very low, but they have the highest concentration of carbonyl compounds. Quantitative analysis of the main odorants followed by the determination of aroma index allow us elucidate the aroma of these varieties. On the basis of their odour description and odour threshold, the most powerful odorants of Tinta Negra Mole wines were tentatively established.

Keywords: Wine; Tinta Negra Mole variety; Aroma compounds; Odorants

Characterisation of volatile fraction of monovarietal wines: Influence of winemaking practices by Z. Piñeiro; R. Natera; R. Castro; M. Palma; B. Puertas; C.G. Barroso (pp. 165-172).

Twenty-three monovarietal wines from 13 white and red grapes have been analysed by solid phase extraction–gas chromatography and solid phase micro-extraction–gas chromatography. The content of some varietal terpenic and fermentative volatile compounds was determined. The 13 studied varieties were characterised as well as the influence of winemaking practices like cold soaking and addition of glycosidase enzymes.Terpenic compounds showed a high variability between the 13 varieties which allows for its characterisation. The winemaking practices devoted to increase the aromatic properties of wines produced different results depending on the variety of grape. For example, cold soaking produced very different results if applied on Palomino fino variety or on Traminer variety, whereas for Viura variety few changes were found.Regarding the volatile compounds generated during the alcoholic fermentation, its relative amount is clearly related to the kind of fermentation process carried out, and particularly according to whether maceration of the solid parts has or has not been done during the process. In the case of vinification without maceration, there is a relative increase of fatty acids and their ethyl esters, whereas in vinification with maceration, the ethyl esters of lactic, acetic and succinic acids are the compounds that are relatively more abundant. Additionally, none of these compounds was affected by the techniques applied to increase varietal aroma, i.e. cold soaking, and addition of glycosidase enzymes showed very low influence in the levels of this kind of compounds.

Keywords: Wine; Grape; Aroma; Terpenic compounds; Varietal aroma; Fermentation

Influence of winemaking techniques on aroma precursors by M. Esti; P. Tamborra (pp. 173-179).

Nero d’Avola (a red grape) and Fiano (a white grape), traditional not aromatic vines from south Italy, have been subjected to different microvinification processes for the purpose of appraising the effects of technology on the aroma precursor composition (both qualitative and quantitative).The volatile compounds derived from glycosidic precursors of wine odorants have been determined through GC–MS analysis of the extracts, after sample purification followed by enzymatic and acid hydrolysis. The results of the enzymatic and chemical hydrolysis of odorant precursors found in the wines produced from Fiano and Nero d’Avola grapes have highlighted the fact that the terpenic, C₁₃-norisoprenoidic, and benzenoidic qualitative compositions of the different wines, after 12 month storage, are very similar to those determined for the grapes. Nero d’Avola is a variety that synthesizes only a limited quantity of α-terpineol and of its derivatives. Fiano grapes and wines, on the other hand, showed the remarkable presence of α-terpineol, and its derivatives but the absence of some benzenoids and hydroxycitronellol. Moreover, cold skin maceration of Fiano grapes, before settling and alcoholic fermentation, greatly increased the aroma precursors of wines. Only some of the glycosidic precursor ratios, among all those considered suitable for grape characterization, remained identical in wine compared to those of the corresponding grape, since they had not been affected by the winemaking technology adopted.

Keywords: Glycoconjugate; Monoterpene; C; ₁₃; -norisoprenoid; Benzenoid; Nero d’Avola; Fiano

Quantitative gas chromatography–olfactometry and chemical quantitative study of the aroma of four Madeira wines by Eva Campo; Vicente Ferreira; Ana Escudero; José C. Marqués; Juan Cacho (pp. 180-187).

The aroma profile of four Madeira wines from the most emblematic grape varieties, Malvazia, Boal, Verdelho and Sercial, has been studied by sensory analysis, quantitative gas chromatography–olfactometry (GC–O) and gas chromatography–mass spectrometry (GC–MS). The aroma of the wines was characterized as candy, nutty, maderized, toasty, lacquer and dried fruit. The GC–O was carried out on extracts obtained by a dynamic headspace technique. The GC–O profiles of Madeira wines were compared to the GC–O profiles obtained from three young white monovarietal wines made with the same varieties so that the identification of odorants related to the particular process of elaboration of Madeira wines was possible. The aroma profile of Madeira wines was extremely complex, and was rich in sotolon, phenylacetaldehyde, wood extractable aromas, and lacked of the most important varietal aromas, such as linalool, 3-mercaptohexyl acetate and methoxypyrazines. A large number of potentially important and unknown odorants, most of them specific to Madeira wines, was also detected by GC–O. The GC–MS results confirmed most of the results of the GC–O study, which suggests that the proposed GC–O strategy is a useful tool for screening the presence of active odorants in wine.

Keywords: Madeira; Grape; Gas chromatography–olfactometry; Quantitative analysis; Sotolon; Phenylacetaldehyde

Changes in volatile composition of Madeira wines during their oxidative ageing by J.S. Câmara; M.A. Alves; J.C. Marques (pp. 188-197).

The influence of the age in the volatile composition of Madeira wines made with Boal, Malvazia, Sercial and Verdelho varieties and aged in oak barrel during 1, 11 and 25 years old was been studied. For this purpose, the evolution of volatile compounds: higher alcohols, ethyl esters, fatty acids, furan compounds, enolic compounds, γ-lactones, dioxanes and dioxolanes, of the four most utilised varieties were determined using liquid–liquid extraction with dichloromeihane. Octan-3-ol was used as internal standard. The wines made with these varieties showed great differences in sugar content and small variations on pH and alcoholic degree.The results show that during ageing, the concentration of fatty acids ethyl esters, acetates and fatty acids decrease significantly contrarily to the great increase of ethyl esters of diprotic acids. There is a strong correlation between sotolon, 2-furfural, 5-methyl-2-furfural, 5-hydroxymethyl-2-furfural and 5-ethoxymethyl-2-furfural with wine ageing. These findings indicate that these compounds can be used as ageing wine markers. Among the molecules studied, sotolon [3-hydroxy-4,5-dimethyl-2(5H)-furanone] was one of the few molecules present in concentrations above the perception threshold in Madeira wines. 5-Eihoxymethyl-2-furfural formed from 5-hydroxymethyl-2-furfural and 2-furfural, derived from sugars, are also involved in the aroma of sweet fortified white wines aged in oxidative conditions. The sensory properties change significantly after long periods of conservation.

Keywords: Volatile compounds; Wine aroma; Ageing; Oxidation; Madeira wines

Evolution of oak-related volatile compounds in a Spanish red wine during 2 years bottled, after aging in barrels made of Spanish, French and American oak wood by Brígida Fernández de Simón; Estrella Cadahía; Teresa Hernández; Isabel Estrella (pp. 198-203).

A red Rioja wine aged for 21 months in barrels made of Spanish oak wood ( Quercus spp.), was stored in bottles for 24 months at wine cellar. The evolution of some volatile compounds during their storage time in bottles was studied and compared with the evolution of the same wine, but aged in barrels made of French oak ( Q. robur, Q. petraea) and American oak ( Q. alba). Although the volatile composition of wine underwent an evolution during bottle aging, this was carried out in such a way that the most important characteristics spread into wine from wood, remained until the end of bottle aging, or at least, for the 24 months of our research. Therefore, wines with different characteristics were obtained from the same wine, after 21 months of aging in barrels and 24 months of aging in bottle, depending on the kind of wood used in oxidative aging.

Keywords: Wine; Aging; Volatile compounds; Quercus; spp.; Oak wood

Headspace-SPME applied to varietal volatile components evolution during Vitis vinifera L. cv. ‘Baga’ ripening by Elisabete Coelho; Sílvia M. Rocha; Ivonne Delgadillo; Manuel A. Coimbra (pp. 204-214).

Grape berries of Vitis vinifera L. cv. Baga were collected during 7 weeks since half- véraison to a post-maturation stage. Two vineyards in different locations in Bairrada Appellation, Pedralvites (Ped) and Colégio (Col) were used. The free varietal and pre-fermentative related volatile compounds that arise in the first 2h after crushing the grapes were followed by Headspace-Solid Phase Microextraction (HS-SPME). Twenty-three sesquiterpenoids, 13 monoterpenoids, 6 norisoprenoids, 2 aromatic alcohols, and 1 diterpenoid were detected in both vineyards. However, 40 sesquiterpenoids and 10 norisoprenoids were detected in Ped. The maximum gas chromatographic peak area was reached at the maturity, and remained constant until post-maturation. At maturity sesquiterpenoids represented 56% and 80% of the total varietal GC peak area in Col and Ped, respectively, which was the group with higher number of constituent varietal compounds. The results obtained indicated a high number of sesquiterpenoid compounds, namely (+)-cycloisosativene, γ-elemene, α-ylangene, β-bourbonene, β-cubenene, β-caryophyllene, 3,7-guaiadiene, (−)-isoledene, (+)-aromadendrene, α-amorphene, (−)-δ-selinene, germacrene D, epizonarene, β-cadinene, γ-cadinene, δ-cadinene, α-muurolene, α-calacorene in ‘Baga’ ripe grapes, which allows to suggest that this variety is a potential source of sesquiterpenoids.

Keywords: Sesquiterpenoids; Vitis vinifera; L.; ‘Baga’; Varietal volatiles; Ripening; Headspace-Solid Phase Microextraction

Behaviour and characterisation of the colour during red wine making and maturation by Enrique García-Puente Rivas; Cristina Alcalde-Eon; Celestino Santos-Buelga; Julián C. Rivas-Gonzalo; M. Teresa Escribano-Bailón (pp. 215-222).

During winemaking and ageing, the colour of red wine evolves from the initial purple-red hues of young red wines towards more red-orange ones. Simultaneously, a modification in the pigment profile takes place. The stability of pigments isolated from red wine has been usually studied in model solutions. However, studies carried out on wines are scarce. The objective of this work was to analyse the changes in composition, colour and stability in relation to pH and SO₂ that take place in a red wine during winemaking and maturation. For this purpose, samples of red wine from Tempranillo grapes were collected periodically during 18 months and submitted to chromatic analysis using the CIELAB and CIELUV colour spaces and high performance liquid chromatography–diode array detection coupled to a mass spectrometer analysis (HPLC–DAD–MS). The results obtained showed the existence of processes capable of causing quantitative and qualitative changes in the colouring material of the wine. These changes, in spite of the relatively short time considered, are sufficient to cause changes of colour that can be perceived by the human eye and lead to the formation of pigments that are more stable against pH and against bleaching by SO₂ than the original anthocyanins.

Keywords: Wine; Colour; CIELAB; Anthocyanins; Anthocyanin-derived pigments

Redox potential evolution during red wine aging in alternative systems by M. del �?lamo; I. Nevares; L.M. Cárcel (pp. 223-228).

The variations of redox potential ( E_H) reflect the oxidation–reduction reactions happening to the wine throughout its aging period. The present study shows the evolution of redox potential in a red wine during aging in different systems (barrels, oak chips and oak staves). All of them have undergone by the same aging conditions carried out in a wine cellar participating the Cigales Appellation of Origin Program (Spain). The monitoring of E_H, basic parameters, color parameters and phenolic compounds explains their modifications, as well as the correlations between the E_H and these parameters with aging time. The redox potential is a useful discriminant variable to differentiate wine aging systems.

Keywords: Aging system; Barrel; Chips; Polyphenols; Red wines; Redox potential; Staves; Online

Monitoring of the ageing of red wines in oak barrels by means of an hybrid electronic tongue by Vicente Parra; �?lvaro A. Arrieta; José A. Fernández-Escudero; Montserrat �?ñiguez; José Antonio de Saja; María Luz Rodríguez-Méndez (pp. 229-237).

A novel hybrid sensor array based on voltammetric electrodes has been successfully applied to monitor the ageing of red wines and to discriminate wine samples aged in oak barrels of different characteristics (wood origin and the toasting level). The hybrid sensor array is formed by three families of chemically modified electrodes, including polypyrrole sensors doped with a range of counterions, carbon paste electrodes modified with metallophthalocyanine complexes and finally, carbon paste electrodes modified with perylene imide derivatives. The diversity of the sensing materials has allowed obtaining a high cross-selectivity in the responses of the sensors forming the array. This voltammetric signals provided by the sensors have been used as data source for a principal component analysis (PCA) and soft independent modelling of class analogy (SIMCA) to confirm the high capability of discrimination and classification of the e-tongue.The array of voltammetric sensors has been able to follow the changes experienced by red wines aged in oak barrels after 3 and 6 months of ageing. In addition, the differences caused by oak barrels of different origins (French, Lithuanian and American) and toasting levels have also been discriminated.

Keywords: Electronic tongue; Sensor array; Carbon paste electrode; Polypyrrole; Phthalocyanine; Perylene; Wine; Oak barrel

Changes in the detailed pigment composition of red wine during maturity and ageing by Cristina Alcalde-Eon; María Teresa Escribano-Bailón; Celestino Santos-Buelga; Julián C. Rivas-Gonzalo (pp. 238-254).

The qualitative and quantitative changes produced, during maturation and ageing, in the pigment composition of a red wine made from Vitis vinifera cv Tempranillo grapes have been studied. In order to determine the detailed composition of the main pigment families involved in the colour changes, a fractionation of the samples has been carried out. One-hundred and twenty-nine different compounds have been identified and their evolutions with wine age have also been established. The data obtained in the analyses of the fractions by high performance liquid chromatography–diode array detection coupled to mass spectrometry (HPLC–DAD–MS) have been used in pigment identification. In order to confirm the identity of some of these compounds, their syntheses have also been carried out. As far as we know, compounds originated by acylation of the monoglucosides of the anthocyanins with lactic acid as well as 3,7-diglucosides of anthocyanins have, among others, been reported here for the first time. The moments of appearance and disappearance of all the detected compounds have also been established as well as the changes in the levels of the different pigment families and subfamilies originated as a consequence of maturation and ageing of the wine in barrels or in bottles. As wine became older, the percentages of anthocyanins decreased slightly, whereas that of the anthocyanin-derived pigments increased and, above all, compounds providing the wine with orange hues (pyranoanthocyanins). In the last sample, they represented 70% of the anthocyanin-derived pigments. On the contrary, the percentage of compounds providing bluish hues to the wine, direct and acetaldehyde-mediated flavanol–anthocyanin condensation products, decreased. This change in the nature of the anthocyanin-derived pigment would explain the colour change observed in the samples, from purple towards orange hues.

Keywords: Wine colour; Anthocyanins; Anthocyanin-derived pigments; Pyranoanthocyanins; Anthocyanin–flavanol condensation products; Ageing

Wine aging in bottle from artificial systems (staves and chips) and oak woods by M. Del Alamo Sanza; I. Nevares Domínguez (pp. 255-263).

A red wine evolution, Cigales appellation of origin, was studied during its storage in bottle after have been aged with three different systems (barrels, chips and staves) made of different kinds of oak wood (American, French and Hungarian). The studied variables were monoglucosides of delphidin, cyanindin, peonidin, petunidin and malvidin, as acetic and p-coumaric esters, as well as vitisín A and malvidin-3- O-caffeylglucoside. During the aging in bottle the wine previously treated in contact with oak chips and staves underwent a quicker aging, their loss of anthocyanins was also quicker and they suffered a higher number of polymerisations than the wine aged in barrels.The analysis of the results allowed to know wine evolution accordingly to the aging system and the botanical species of wood used. Wine treated with staves had a quicker evolution in bottle than wines treated with the other two systems because there was a higher loss of anthocyanins. On the other hand, wine treated with French oak wood suffered a slightly lower loss of anthocyanins than those aged with Hungarian and American oak wood.

Keywords: Aging; Anthocyanin; Barrels; Bottle; Chips; Red wine; Staves

Flavour and odour profile modifications during the first five years of Lourinhã brandy maturation on different wooden barrels by Ilda Caldeira; A.M. Mateus; A.P. Belchior (pp. 264-273).

The same Portuguese brandy from the region of Lourinhã was used to fill 63 different wooden barrels. These barrels were made according to a two factorial experiment (7 woods×3 toasting levels×3 replicates). Samples of the brandies were taken after two, three, four and five years of maturation time.The flavour and odour profile of these 63 samples were evaluated each year by a panel of judges, previously selected and trained, using a structured scale for 20 attributes.The results obtained showed a significant effect of maturation time on several sensory attributes, namely alcohol, fruity, vanilla, woody, burned/toasted, dried fruits, smoke, tails, glue, smooth, caoutchouc, burning, astringency, bitterness, body, flavour complexity, retronasal aroma and flavour persistence. The panel also evaluated the overall quality of the brandies, which was also strongly influenced by the maturation time. The intensity of many positive brandy attributes (vanilla, woody, dried fruits, toasted, smoke, body, flavour complexity, retronasal aroma and flavour persistence) increased over the time, as well as the overall quality. Conversely, the intensities of other attributes, such as alcohol, astringency, fruity, glue, bitterness and burning, decreased over the time.In addition, the results showed that the brandies presented significant differences on flavour and odour profile, resulting from different wooden barrels and from different toasting levels of the barrels used for the maturation.

Keywords: Brandies; Maturation; Sensory profile; Chestnut wood; Oak wood; Toasting level

DNA analysis in wines: Development of methods for enhanced extraction and real-time polymerase chain reaction quantification by Federica Savazzini; Lucia Martinelli (pp. 274-282).

Reliable methods for DNA traceability in grapevines and wines is in great demand for protecting areas of declared origin and detecting potential transgenic events. Currently, real-time polymerase chain reaction (PCR) is one of the most promising tools for conducting plant and microorganism DNA assays and detecting genetically modified material. However, in grape, quantitative analysis based on PCR is lagging behind. Moreover, in musts and wines, efficient DNA extraction and amplification need to be developed. In the present research, we compared several DNA extraction procedures on various grape tissues, musts and wines of the Trentino-Südtirol Region. We chose the Vitis vinifera nine-cis-epoxycarotenoid dioxygenase2 (VvNCED2) gene as the referee standard in grape and tested for validation. Using real-time PCR, with VvNCED2 as standard referee, we successfully quantified grape DNA from grape tissues and the residual grape DNA in musts and wines. In addition, we also quantified the S. cerevisae residual DNA in wines. Extracted grape DNA quality and specificity were also verified by co-dominant simple sequence repeat (SSR) markers. SSRs proved that, even 1 year after wine fermentation, we could detect and identify grape DNA that was still suitable for analysis. Our research is the first successful report of efficient DNA extraction, amplification and real-time PCR quantification for aged wines. As a result, we could count on a suitable assay for tracing and monitoring endogenous and exogenous DNA in grape and grape products.

Keywords: Real-time polymerase chain reaction; Grape endogenous gene; DNA quantification; Microsatellite

Detection and identification of grape varieties in must and wine using nuclear and chloroplast microsatellite markers by M.M. Baleiras-Couto; J.E. Eiras-Dias (pp. 283-291).

Characteristics such as production yield, alcoholic level, acidity and anthocyanin levels vary among grape varieties. Since these characteristics are highly correlated with the final wine quality, it is important to be able to detect and correctly identify the grape varieties (cultivars) present in musts and wines. This is particularly relevant in controlling the quality and authenticity of monovarietal wines.In this study, molecular methods based on residual DNA analysis of Vitis vinifera L. cultivars were applied to musts and wines produced from five different cultivars, namely Touriga Franca, Fernão Pires, Tinta Barroca, Tinto Cão and Marselan. Initially, three DNA extraction methods were compared and optimised for the isolation of DNA from must and wine and then six nuclear and two chloroplast microsatellite markers were used to identify single-varietal and blends of two varieties of musts prepared in laboratory and single-varietal wines produced in microvinifications.Preliminary results on multivarietal musts indicated a possible relationship between the proportion of each variety in the mixture and the signal intensity of the alleles obtained in an automatic sequencer, suggesting that it could be possible to quantify the presence of each variety in the mixture.A method for DNA extraction from wine was developed and shown to be adequate for microsatellite amplification. Results have shown for the first time that markers targeting short DNA fragments at chloroplast genomes are useful tools to detect grapevine DNA in wines. This suggests that it may be possible to apply these techniques in controlling wine quality and origin certification, as well as in detecting wine falsification.

Keywords: Vitis vinifera; L.; Wine characterisation; Grapevine identification; DNA markers; Nuclear simple sequence repeats (SSR); Chloroplast simple sequence repeats (cpSSR)

Investigation of variety, typicality and vintage of French and German wines using front-face fluorescence spectroscopy by É. Dufour; A. Letort; A. Laguet; A. Lebecque; J.N. Serra (pp. 292-299).

The potential of front-face fluorescence spectroscopy combined with chemometric methods was investigated for discriminating different wines according to variety, typicality and vintage. A total of 120 wines produced in France and Germany were investigated. Emission (275–450nm) and excitation (250–350nm) fluorescence spectra of phenolic compounds were recorded directly on the wine samples using a front-face fluorescence accessory. The emission spectra were characterised by a maximum at 376nm and a shoulder at 315nm and the excitation spectra showed two peaks located at about 260 and 320nm. The shape of the spectra changed with wine samples, varying mainly in the maximum/shoulder intensity. Wine samples were evaluated using principal component analysis (PCA) and classified by factorial discriminant analysis (FDA). PCA performed on the whole collection of excitation spectra allowed a good discrimination between French and German wines. Using FDA, correct classification of typical and non-typical Beaujolais amounting to 95% was observed for the emission fluorescence data set. These results showed that fluorescence spectroscopic technique may provide useful fingerprints and mainly allow the identification of wines according to variety and typicality.

Keywords: Polyphenol; Tannin; Fluorescence; Chemometry; Authentication

C₆-alcohols as varietal markers for assessment of wine origin by José M. Oliveira; Marta Faria; Filomena Sá; Filipa Barros; Isabel M. Araújo (pp. 300-309).

A significant part of the compounds present in wines having six carbon atoms, the C₆-compounds, derive from grape polyunsaturated fatty acids (primarily originated from membrane lipids), namely linoleic and α-linolenic acids, through a cascade of enzymatic reactions. This biochemical pathway yield C₆-aldehydes, which are subsequently reduced to C₆-alcohols, which can, in turn, be esterified to produce esters. As the C₆-compounds derive from varietal precursors, they could hypothetically contribute to judge wine origin and affiliation. In this way, two C₆-alcohols, ( E)-3-hexenol and ( Z)-3-hexenol, have been referred as the most important because its ratio can act as an indicator of the variety of origin.This study presents the results, concerning the concentration of the three main C₆-alcohols, 1-hexanol, ( E)-3-hexenol and ( Z)-3-hexenol, as well as ratios between them, for 43 monovarietal wines from Vinhos Verdes demarcated region, belonging to six white – Alvarinho (8), Arinto (1), Avesso (9), Azal (1), Loureiro (17) and Trajadura (4) – and three red – Amaral (1), Borraçal (1) and Vinhão (1) – grape varieties. Wines were produced at experimental scale using slightly different winemaking practices and representing various terroirs and vintages, being analyzed after different conservation periods.The results showed that ( E)-3-hexenol/( Z)-3-hexenol ratio clearly discriminates Loureiro wines from those of Alvarinho, Avesso and Trajadura. Moreover, 1-hexanol/( E)-3-hexenol and 1-hexanol/( Z)-3-hexenol ratios may also be able to discriminate Vinhos Verdes monovarietal wines, and can act on a second level differentiation. The remaining monovarietal wines produced results which may be observed as indicative, since only one sample of each was analysed.

Keywords: C; ₆; -compounds; 3-Hexenol; 1-Hexanol; Wine discrimination; Vinhos Verdes

Determination of total chloroanisoles in different kinds of cork stoppers by M. Riu; M. Mestres; O. Busto; J. Guasch (pp. 310-314).

This paper deals with the application of a new headspace solid-phase microextraction (HS-SPME) method developed in order to analyse the total amount of chloroanisoles in different kinds of cork stoppers (natural, agglomerated and sparkling wine stoppers). This parameter must be determined because these compounds can migrate from cork to wine giving it unpleasant musty taint and, therefore, undermine its organoleptic quality. Unlike the releasable amount of chloroanisoles, the total amount of chloroanisoles has no dependence of the method used for its determination so it can be used as a quality control parameter. The application of this method allowed the simultaneous determination of 2,4-dichloroanisole, 2,6-dichloroanisole, 2,4,6-trichloroanisole, 2,3,4,6-tetrachloroanisole and 2,3,4,5,6-pentachloroanisole.

Keywords: Cork; Chloroanisoles; Solid-phase microextraction; Gas chromatography; Total amount of chloroanisoles; Quality control

Quality evaluation of cork from Quercus suber L. by the electronic tongue by Alisa Rudnitskaya; Ivonne Delgadillo; Silvia M. Rocha; Ana-Maria Costa; Andrey Legin (pp. 315-318).

The aim of the present study was application of the electronic tongue multisensor system (ET) for discrimination of cork both standard and Mancha Amarela (Yellow spot). Extracts of cork in 10% ethanol water solutions were analysed. Two sets of cork samples that included both standard (S) and Mancha Amarela (MA) cork samples from two different factories were studied. It was found that ET could reliably distinguish extracts made from S and MA cork regardless samples’ origin. ET could predict total phenols’ content with average precision of 9% when calibrated using reference data obtained by Folin–Ciocalteu method. The ET was demonstrated to be promising tool for the cork quality evaluation.Difference in composition between S and MA cork was studied by ET. The largest difference in concentration between two types of cork extracts was found for gallic and protocatechuic acids.

Keywords: Cork; Yellow spot-Mancha Amarela; Electronic tongue; Phenolic components

Combining mass spectrometry based electronic nose, visible–near infrared spectroscopy and chemometrics to assess the sensory properties of Australian Riesling wines by Daniel Cozzolino; Heather E. Smyth; Kate A. Lattey; Wies Cynkar; Les Janik; Robert G. Dambergs; I. Leigh Francis; Mark Gishen (pp. 319-324).

The combination of mass spectrometry (MS) based electronic nose (eNose) with visible (VIS) and near infrared spectroscopy (NIR) was explored as an objective tool to measure sensory attributes in commercial Riesling wines grown in Australia. Calibration models were developed between instrumental data and sensory scores using partial least squares (PLS) regression with full cross validation. Good correlations ( r>0.70, root mean square standard error in cross validation (RMSECV): 0.66) were found for developed and floral; intermediate (0.70> r>0.60, RMSECV: 0.84 and 0.63) for tropical and low (r<0.50, RMSECV: 0.98) for green characters measured by a sensory panel and the combination of both techniques. The results suggested that data from instrumental techniques coupled with chemometrics might be related with sensory scores measured by a trained panel. The study is considered a starting point in order to evaluate useful sources of information generated by different instrumental techniques with the objective to select combination of sensors for specific wine quality attributes.

Keywords: Mass spectrometry electronic nose; Near infrared spectroscopy; Chemometrics; Riesling; Sensory attributes; Partial least squares

Stable isotope analysis in grape products:¹³C-based internal standardisation methods to improve the detection of some types of adulterations by G. Versini; F. Camin; M. Ramponi; E. Dellacassa (pp. 325-330).

Correlations between δ¹³C of total sugars, heterosides and the sugar fraction from heterosides were investigated, as well as those between amino acid fractions isolated from musts and relevant wines and, in this frame, between alcohol and amino acids in wine. The target of this study was to find internal standardisation elements which could reduce the uncertainty limit to detect possible adulteration in wine, such as addition of C4-plant sugar.As for the investigation on heterosides, isolated both from experimental and industrial musts and relevant concentrated rectified musts, a highly significant linear correlation between δ¹³C value of sugar from heterosides and total sugar (δ¹³C sugar=−1.57+0.87×δ¹³C heterosides sugar; r=0.88; N=26) was found. It is worth mentioning the mean¹³C difference of about 1.7‰ between free glucose and glucose from heterosides, as well as the resulted predictability of at least 10% of added C4-plant sugar.As for the correlation between δ¹³C of the amino acid fraction of musts and relevant wines referring to 70 mostly microvinified white and red renowned varietal grapes, a highly significant linear relationship was obtained. Considering δ¹³C values of alcohol and the amino acid fraction of wines, measured in the same samples and in more than 50 wines from the official Italian isotope data-bank, it resulted that the¹³C content of alcohol cannot be higher than that of amino acids. This tendency was observed also in foreign wines as, e.g. in Argentinean products, while for the Uruguayan ones, where addition of C4-plant sugar was allowed until 10% of total alcohol, most samples showed an inverse isotopic situation confirming the validity and sensitivity of this approach.

Keywords: Abbreviations; CRM; concentrated rectified must; ¹; H NMR; ¹; H nuclear magnetic resonance; HPAE-PAD; high pressure anionic exchange–pulse amperometric detector; HPLC; high pressure liquid chromatography; HRGC; high resolution gas chromatography; IRMS; isotope ratio mass spectrometry; PVPP; polyvinyl-polypyrrolidone and pulsed; SNIF; ²; H NMR; site-specific natural isotopic fractionation; ²; H nuclear magnetic resonance; V-PDB; Vienna-Pee Dee Belemnite¹³; C/; ¹²; C analysis; Heterosides; Amino acids; Internal standardisation relationships; C4-plant sugar addition

Chromatic characterisation of three consecutive vintages of Vitis vinifera red wine by I. Esparza; C. Santamaría; J.M. Fernández (pp. 331-337).

Both standard and CIELAB colour parameters have been evaluated for three consecutive vintages of Vitis vinifera (Tempranillo variety). A simple and straightforward relationship between CI and L^* has been derived. Dilution effects on colour for samples under fermentation and for several commercial wines have been studied under pH controlled conditions. Disruptions of equilibria responsible for colour at the typical wavelengths upon addition of iron have been assessed.

Keywords: Wine colour; Red wine; CIELAB; Colour Intensity; Dilution study; Iron

Monitoring a commercial fermentation with proton nuclear magnetic resonance spectroscopy with the aid of chemometrics by Shona Clark; Neil William Barnett; Mike Adams; Iain Bruce Cook; Gail Anne Dyson; Geoff Johnston (pp. 338-345).

Proton nuclear magnetic resonance spectroscopy (NMR) has shown the potential for being a valuable tool in monitoring a commercial fermentation. In this preliminary study, a suite of organic analytes including ethanol, fructose, glucose, methanol, glycerol, malic acid, tartaric acid, succinic acid, acetic acid and lactic acid were simultaneously determined during the fermentation. Data collection and analysis using chemometric algorithms aided the understanding of key processes including the effects of seeding a wine with bacteria for malo-lactic fermentation.

Keywords: Nuclear magnetic resonance; Fermentation; Chemometrics; Organic acids; Ethanol and sugars

¹H NMR metabolite fingerprints of grape berry: Comparison of vintage and soil effects in Bordeaux grapevine growing areas by Giuliano E. Pereira; Jean-Pierre Gaudillere; Cornelius van Leeuwen; Ghislaine Hilbert; Mickaël Maucourt; Catherine Deborde; Annick Moing; Dominique Rolin (pp. 346-352).

Many variables such as total soluble sugars, total titratable acidity, nitrogen and phenolic compounds balance, contribute to describe grape quality. They vary strongly with genetic (cultivars) and environmental (climate, soil, and cultural practices together referred to as “terroir�?, and vintage) factors. The aim of this work was to determine changes in metabolite fingerprints of grape berry skins of ‘Merlot noir’, ‘Carbernet franc’ or ‘Cabernet Sauvignon’ cultivars harvested in 2002, 2003 and 2004 from five geographical locations in Bordeaux (south-west of France) to better understand the factors influencing the grape berry composition.¹H NMR spectroscopy was used to determine metabolic profiles of skin extracts of berries harvested at maturity. Before analysis by multivariate statistical methods, spectral data were reduced (sum of intensities over 0.04ppm spectral domains) and normalized to generate 183 variables describing the entire spectra. Principal component analysis (PCA) and partial least squares (PLS) chemometric methods were applied to describe sample variability and show clustering of samples. These chemometric methods gave a good separation of samples according to vintages. PLS allowed us to pinpoint spectral domains corresponding to metabolites contributing to the differences between vintages, including sugars (glucose, fructose and sucrose), organic acids (tartaric, malic, citric and succinic acids), and amino acids (proline, arginine, GABA, valine, alanine, leucine and isoleucine). Conversely, soils classified according to their capacity to provide water to the vines, could not be discriminated by¹H NMR metabolic fingerprinting of the berries. The vintage effect on grape metabolic profiles prevailed over the soil effect. This emphasizes the fact that a typical vineyard is defined by its most frequent climatic traits such as the seasonal sum of temperatures and water balance.

Keywords: Grape berry; Vintage; Terroir; ¹; H NMR spectroscopy; Multivariate statistical analysis; Principal component analysis; Partial least squares

Metabolism of linalool and substrate analogs in grape berry mesocarp of Vitis vinifera L. cv. Morio Muscat: demonstration of stereoselective oxygenation and glycosylation by Fang Luan; Armin Mosandl; Andreas Degenhardt; Michaela Gubesch; Matthias Wüst (pp. 353-364).

The oxidative metabolism of linalool in Vitis vinifera L. cv. Morio Muscat has been investigated by in vivo feeding experiments using the regioselectively stable isotope-labelled substrates d₅-(3 R/ S)-linalool, d₆-(3 R)-linalool, d₂,¹⁸O-(3 R/ S)-linalool and the substrate analog (3 R)-linalyl methyl ether. The enantiomeric and diastereoisomeric ratios of the metabolites were determined by means of enantioselective multidimensional gas chromatography–mass spectrometry. Stereoselective transformation to diendiol II and the furanoid and pyranoid linalool oxides could be demonstrated. Other metabolites like diendiol I, hotrienol and 8-hydroxy linalool were detectable as well. The studies indicate that the corresponding metabolites were efficiently glycoconjugated. Time course studies including the determination of conversion rates revealed that the activity of these secondary transformations is dependent on the ripening stage. The mass spectrometric analysis of the labelled linalool oxides derived from mixed labelled d₂,¹⁸O-linalool and the stereoselective anaylsis of the metabolite 6-methoxy-2,6-dimethyloct-7-en-2,3-diol, which is derived from the substrate analog (3 R)-linalyl methyl ether, give evidence that the furanoid linalool oxides are generated via two different reaction pathways.

Keywords: Monoterpenes; Oxidative metabolism; Enantioselective multidimensional gas chromatography–mass spectrometry (enantio-MDGC–MS)

Stability of sotolon in acidic and basic aqueous solutions by L. Dagan; R. Schneider; J.P. Lepoutre; R. Baumes (pp. 365-374).

Up to 50°C, sotolon1 slightly decomposed to acetoin5 in aqueous sodium hydroxide solution under nitrogen and in the absence of light, and this degradation was irreversible, but at 80°C it was rapidly decomposed according to two other reaction pathways. The first one (kinetic route) was new: it gave rise to the novel 3-methyl-2-oxopent-3-enoic acid11 by opening of the lactone cycle. The second one (thermodynamic route) was the reverse of the formation of1 from ethanal9 and 2-oxobutyric acid8, giving also propionic acid10 by decarbonylation of8. As shown in dilute potassium hydroxide added with potassium fluoride, the initial addition of hydroxide ion to1 distinguished between the processes at 50 and 80°C. At 50°C, the ion added to the soft electrophilic carbon of the conjugated lactone (1,4-addition), whereas at 80°C, it added to the hard electrophilic carbonyl carbon (1,2-addition). Under acidic conditions,11 was easily cyclised back to racemic1, whereas1 formation from ethanal and 2-oxobutyric acid was very slow and unspecific, as reported previously. Furthermore, 5,5,5[²H₃]-3-methyl-2-oxopent-3-enoic acidd₃-11 was produced from1, when its degradation was carried out in sodium deuteroxide at 80°C. Its cyclisation in deuterated acid solution gave rise to 3-hydroxy-4-methyl-5-[²H₃])-methylfuran-2(5H)-oned₃-1, which was shown to be chemically and isotopically stable in strong acidic conditions.d₃-11 was used as internal standard in a new stable isotope dilution assay method to quantitate1 in wine. As1 was shown to decompose slowly to the above degradation products in dilute sodium carbonate at room temperature, but faster in the presence of cupric ion (mainly oxidation to5), a purification procedure using the favoured partition of hydrophilic1 in dilute aqueous acid allowed to obtain extracts suitable to quantitate specifically1. This method allowed the quantitative determination of1 in Petit and Gros Manseng dry and sweet Jurançon wines (9–24 years ageing), with levels below its olfactive perception threshold (15μgL⁻¹).

Keywords: Sotolon; 3-Methyl-2-oxopent-3-enoic acid; Degradation; Wine; Stable isotope dilution assay; Manseng

Ethyl caffeoate: Liquid chromatography–tandem mass spectrometric analysis in Verdicchio wine and effects on hepatic stellate cells and intracellular peroxidation by Natale G. Frega; Emanuele Boselli; Emanuele Bendia; Massimo Minardi; Antonio Benedetti (pp. 375-381).

Analysis of Verdicchio (a white wine produced in Italy) with high performance liquid chromatography–tandem mass spectrometry (HPLC–ESI-MS–MS) showed a concentration of ethyl caffeoate (CfE), a natural phenolic substance, of 67.3μmolL⁻¹; CfE was then purified from wine with preparative reversed phase HPLC and tested on rat hepatic stellate cell cultures (HSC). HSC proliferation induced by iron(III) nitrilotriacetate (FeNTA), a pro-oxidant agent (absorbance=0.68±0.03 versus 0.32±0.02 in controls), was significantly inhibited by CfE starting at a concentration of 10μmolL⁻¹ (absorbance=0.45±0.01), with a maximal effect at 20μmolL⁻¹ (absorbance=0.32±0.02). The formation of intracellular hydroperoxides induced by FeNTA was significantly reduced by CfE starting at a concentration of 10μmolL⁻¹ and completely prevented by CfE at a concentration of 20μmolL⁻¹. The increase in α1(I) procollagen mRNA synthesis induced by FeNTA was inhibited by CfE starting at a concentration of 10μmolL⁻¹ and almost completely at a concentration of 20μmolL⁻¹. Thus, Verdicchio wine is a remarkable source (about seven-fold the bioactive concentration) of CfE, an interesting drug to be tested as an antifibrotic agent.

Keywords: Ethyl caffeoate; Verdicchio wine; Mass spectrometry; Antioxidants; Hepatic stellate cells; Chromatography; Liver fibrosis

Stilbenes: Quantitative extraction from grape skins, contribution of grape solids to wine and variation during wine maturation by Baoshan Sun; Ana M. Ribes; M. Conceição Leandro; A. Pedro Belchior; M. Isabel Spranger (pp. 382-390).

With the objective of studying the relationship between stilbenic composition in grape skins and that in corresponding red wine, we have firstly optimized a method for quantitative extraction and purification of stilbenes from grape skins, used for posterior HPLC analysis. Stilbene levels in grape skins of three Vitis vinifera varieties (Castelão, Syrah and Tinta Roriz) and in their corresponding wines made by traditional winemaking technologies were analyzed. Stilbene composition in grape skin varied considerably, depending on the grapevine variety. The contents of trans-resveratrol in grape skins of the three varieties were not significantly different. However, their contents of trans-piceid on dry skin were very significantly different (67.24, 10.43 and 11.57mgkg⁻¹, respectively). Moreover, cis-piceid, which was absent in Syrah and Tinta Roriz grape skins, was detected in Castelão grape skin with an important amount (58.9mgkg⁻¹ on dry skin). The high concentrations of stilbenes were found in stem tissue (cv. Castelão): 145.52mgkg⁻¹ of trans-resveratrol, 61.43mgkg⁻¹ of trans-piceid and 143.85mgkg⁻¹ of cis-piceid (on dry stem), while only trans-resveratrol was quantified in the grape seeds with a low concentration (6.8mgkg⁻¹ on dry seed). Simulated maceration of each solid part of grapes using model wine solution indicated that both stem and seed have little contribution of stilbenes to wines while skins are the major source of these compounds in wine. On the other hand, cis-piceid was not detected in the corresponding wines made by these grapevine varieties, but its isomer trans-piceid was detected in an important concentration, suggesting that cis-piceid, once extracted, isomerized to its trans-form. The evolution of these compounds during the elaboration of wines (from the end of alcoholic fermentation until second racking, i.e., just before bottling) showed that in general, trans-resveratrol concentration decreases gradually, trans-piceid kept constant, while cis-resveratrol increases slightly. The important finding of this work is that there was a significant correlation between the concentration of total piceid in grape skins and that in the respective wine. On the contrary, no such relationship was found for resveratrol.

Keywords: Stilbenes; Resveratrol; Piceid; Grape; Wine

Oxygen measures and consumption in must and wine by Angela Silva; Milena Lambri (pp. 391-395).

The aim of this work is to point out a system of oxygen measure on real scale and to estimate the oxygen concentrations in must in red winemaking, in particular during maceration and alcoholic fermentation.The experimental vat had two different controlled aeration systems of must: during pumping-over or during the re-circulation of a fraction of the liquid. For the evaluation of the oxygen dissolved during maceration, the vat was equipped with devices to carry out the measure in the must before the aeration, in the must aerated during the re-circulation, after the pumping-over and in the total mass.The measure of dissolved oxygen were made with an instrument based on the Clark electrode, provided with automatic compensation for the temperature and the salinity.In order to obtain the consumption kinetics, the measures were made in the time range necessary to reach the values determined before aeration.During maceration, the oxygen concentration returns to the initial value in variable times, ranging from few minutes to 20min. In the first 2–3min the oxygen dissolved in the total mass is reduced of 50–60% during the tumultuous fermentation and of the 35–50% at the end of alcoholic fermentation. Subsequently the decrease is gradual and slower and the oxygen concentration was closed to the instrumental detection limit.The consumption kinetics, apart from the mechanism, can be represented by an exponential equation.

Keywords: Oxygen; Red winemaking; Maceration; Alcoholic fermentation; Consumption kinetics

A study on the composition of distillates obtained from smoked marc by Carla Da Porto; Sabrina Moret; Susi Soldera (pp. 396-400).

Fermented marc is the traditional raw material of grappa. The effects of a controlled smoking process applied to fermented marc for 120, 240 and 460min on the composition of the corresponding distillates (spirits at ca. 40%, v/v ethanol) were studied. The smoked marc samples were distilled using a pilot plant and the distillates collected by volume and analysed. Longer was the exposition of the marc samples to the smoking process higher resulted the reduction of ethanol, higher alcohols and ethyl esters. Instead, the volatile phenols and the polycyclic aromatic hydrocarbons (PAHs) increased with the extended of smoking time. Good correlations between the time of smoking and the concentrations of higher alcohols and PAHs have been found. In relation to the content of benzo( a)pyrene, it must be noticed that it was detected always in concentrations which did not exceed the limit of 0.03μgkg⁻¹.

Keywords: Marc; Smoking process; Distillation; Phenols; Polycyclic aromatic hydrocarbons

Chemical characterization of commercial dietary ingredients from Vitis vinifera L. by María Monagas; Ignacio Garrido; Begoña Bartolomé; Carmen Gómez-Cordovés (pp. 401-410).

In recent years a large number of commercial products containing ingredients from winery byproducts have proliferated in the market. However, little research has focused on studying the phenolic composition of these products and there is still no legislation concerning their commercialization. In this work, a wide range of commercial ingredients derived from Vitis vinifera L. grape seeds ( n=16) and skins ( n=4), grape pomace ( n=6) and leaves ( n=3) as well as different production batches of some of them, have been analyzed. Gallic acid, monomeric flavan-3-ols ((+)-catechin, (−)-epicatechin and epicatechin-3- O-gallate), procyanidins dimers (B3, B1, B4, B2, B3′-3- O-gallate), and trimers (T2 and C1) were identified in ingredients derived from grape seeds. These ingredients were comprised of fractions enriched either in monomeric, oligomeric or polymeric flavan-3-ols, with a mean degree of polymerization (mDP) ranging from 2.1 to 11.2. Grape anthocyanins (delphinidin, cyanidin, petunidin, peonidin and malvidin-3-glucosides, -3-(6-acetyl)-glucosides and -3-(6- p-coumaroyl)-glucosides, and peonidin and malvidin-3-(6-caffeoyl)-glucosides) were identified in ingredients derived from non-processed grape skins whereas grape pomace ingredients also contained anthocyanin-derived pigments (the pyruvate, vinylcatechol, vinylepicatechin, vinylphenol and vinylguaiacol derivatives of malvidin-3-glucoside, and malvidin-3-(6-acetyl)-glucoside-vinylepicatechin). In the case of the ingredients of leaves, anthocyanins (delphinidin, cyanidin, petunidin, peonidin and malvidin-3-glucosides and -3-(6- p-coumaroyl)-glucosides, and cyanidin and peonidin-3-(6-acetyl)-glucosides), and non-anthocyanin compounds ( trans-caftaric acid, and the -3- O-galactoside, -glucuronide and -glucoside derivatives of quercetin and kaempherol, and quercetin aglycone) were identified; the relationship between the two types of compounds (anthocyanin/flavonol) differed in the distinct ingredients studied. Despite the large variability found between the different brands of ingredients derived from a particular byproduct, all of the ingredients studied were authentically derived from the V. vinifera L. spp.

Keywords: Reverse phase liquid chromatography–electrospray mass spectrometry; Thiolysis; Flavan-3-ols; Anthocyanins; Anthocyanin-derived pigments; Flavonols; Grape seeds; Grape skins; Leaves

Author Index (pp. 411-414).

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Analytica Chimica Acta (v.563, #1-2)

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Analytica Chimica Acta (v.563, #1-2)