Analysis of grape Vitis vinifera L. DNA in must mixtures and experimental mixed wines using microsatellite markers

Because wine quality highly relies on the varietal composition of the must, the development of methods allowing the authentication of varieties in musts and wines would be of great value as a guarantee of quality. Microsatellite markers have already been applied to the authentication of grape juices (Faria, M. A.; Magalh?es, R.; Ferreira, M. A.; Meredith, C. P.; Ferreira Monteiro, F. J. Agric. Food Chem. 2000, 48, 1096-1100) and to the analysis of experimental wines (Siret, R.; Boursiquot, J. M.; Merle, M. H.; Cabanis, J. C.; This, P. J. Agric Food Chem. 2000, 48, 5035-5040). In the present paper, we accessed the usefulness of this technology for the analysis of must and wine mixtures. The detection limit of DNA mixtures was first estimated on DNA extracted from leaves: 4% of a foreign DNA can be detected. Analysis of must and wine mixtures (Chardonnay B/Clairette B and Syrah N/Grenache N) was performed on experimental fermentations. DNA was extracted along the fermentation process and analyzed using five microsatellite loci. The 70:30 (v/v) mixtures were successfully analyzed until the end of the fermentation. The applications of these results to commercial purposes are discussed.     

Characterization and quantification of grape variety by means of shikimic acid concentration and protein fingerprint in still white wines

Protein profiles, obtained by high-performance capillary electrophoresis (HPCE) on white wines previously dialyzed, combined with shikimic acid concentration and multivariate analysis, were used for the determination of grape variety composition of a still white wine. Six varieties were studied through monovarietal wines elaborated in the laboratory: Chardonnay (24 samples), Chenin (24), Petit Manseng (7), Sauvignon (37), Semillon (24), and Ugni Blanc (9). Homemade mixtures were elaborated from authentic monovarietal wines according to a Plackett-Burman sampling plan. After protein peak area normalization, a matrix was elaborated containing protein results of wines (mixtures and monovarietal). Partial least-squares processing was applied to this matrix allowing the elaboration of a model that provided a varietal quantification precision of around 20% for most of the grape varieties studied. The model was applied to commercial samples from various geographical origins, providing encouraging results for control purposes.     

Toward the authentication of varietal wines by the analysis of grape (Vitis vinifera L.) residual DNA in must and wine using microsatellite markers

In an attempt to develop a technique for the identification of grape cultivars in commercial wines, a method for the extraction of DNA from must and experimental wines was adapted and optimal PCR conditions for the amplification of this DNA were established. DNA was analyzed during the fermentation process for six cultivars (Chardonnay, Clairette blanche, Grenache noir, Merlot, Muscat blanc à petits grains, and Syrah). The extractions were performed on solid parts in suspension as well as on the aqueous fraction. Expected profiles for these cultivars were obtained with DNA extracted from the solid parts during all of the fermentation process and for the wine. The analysis of DNA extracted from aqueous fractions was less reproducible, and microsatellite amplifications were obtained only in the case of Clairette blanche, Merlot, and Syrah wines. Results demonstrate that the purification process is adequate for the analysis but that the DNA concentration represents the main limiting factor. Technical improvements of the method are discussed.     

Determination of free and conjugated indole-3-acetic acid, tryptophan, and tryptophan metabolites in grape must and wine.

Tryptophan (Trp) and its metabolites, especially indole-3-acetic acid (IAA), are considered to be potential precursors of 2-aminoacetophenone (AAP), an aroma compound that causes an "untypical aging off-flavor" (UTA) in Vitis vinifera wines. In this study, RP-HPLC with fluorescence detection was used for the qualitative and quantitative analysis of Trp and Trp metabolites in grape musts and wines to which different viticultural measures had been applied (time of harvest, soil treatment, leaf plucking, vine prune). An alkaline hydrolysis was developed to release bound IAA and Trp. A sensitive and selective determination of different Trp metabolites was achieved after solid phase extraction using a strong anion exchange material. In the examined grape musts, more than 95% of the total IAA was bound either as ester conjugate or as amide conjugate. Free IAA and other Trp metabolites were below the detection limit (<3 microg/L) or could be determined only in traces. Their amounts increased significantly during fermentation, whereas the amount of Trp decreased. It could be shown that the different viticultural measures applied (except the vine prune) as well as the climatic conditions of the vintage exhibited significant influences on the amounts of Trp and Trp metabolites in grape musts or wines.     

Determination of piceid and resveratrol in Spanish wines deriving from Monastrell (Vitis vinifera L.) grape variety

The presence of stilbenes in wine is becoming an important issue due to their claimed relation to a low incidence in coronary diseases and their increasing implication as cancer chemopreventive and neuroprotective agents. Total resveratrol content, quantified as glucoside and aglycone forms of resveratrol, has been determined in a survey of 45 Monastrell monovarietal Spanish red wine types (around 135 wine samples), belonging to Alicante and Bullas appellations. The average between ratio glucoside/aglycone forms of resveratrol in these wines was considerably high, ranging from 82 to 91% of resveratrol in its glycosidic form. This characteristic was observed in a high percentage of the studied wines, which were made under different winemaking procedures, and from different vintages (1995-2002). In addition, wines made using macerative fermentations with double amount of solid parts ("doble pasta") reached the highest levels of total stilbene content expressed as resveratrol equivalent, i.e., 30 mg/L (average of 18.8 mg/L). It can be concluded that high resveratrol glucoside concentration and low free isomer content can be considered characteristics of the Monastrell variety, as it happens to red wines deriving from other varieties grown at warm climates. This fact, also observed for other French and Portuguese red varieties, might play an important role in food habits involving these types of wines.     

Influence of prefermentative treatments to the major volatile compounds of Assyrtiko wines

A study of the volatile fraction of Assyrtiko wines, using gas chromatography coupled with olfactometry, was realized. Twenty-seven volatile compounds were identified as potent odorants, most of them originating from the fermentation process. Quantification of the major volatile compounds was realized developing a rapid analytical method based on fractionation of a 50 mL wine aliquot using C 18-reversed phase adsorbent. After elution of the volatile compounds with pentane-diethyl ether and concentration under nitrogen, the final wine extract was injected in a gas chromatography-flame ionization detection system. The method allows satisfactory determination of more than 15 volatile compounds of wine. The linearity of the method gave a typical r (2) between 0.990 and 0.999, while reproducibility ranged from 5.1 to 12.2% (as relative standard deviation) with 9.5% as the average. The method was applied to wines produced by Assyrtiko grapes (AOC Santorini), for two consecutive years, to compare the effect of skin contact prior to fermentation and the must clarification process. Direct press and skin contact wines were differentiated analytically; however, highly significant differences were not. Inversely, the differences found between direct press/clarified and nonclarified wines were significant. Wines produced by direct press and clarified must presented significantly higher levels of ethylic esters and fusel alcohol acetates but lower fusel alcohol levels, leading probably to more fruity wines. This difference, between clarified and nonclarified grape musts, was not significant in the case of the wines produced by skin contact of Assyrtiko berries. These findings were validated by preference sensory analysis tests.     

Butyltin compounds in Portuguese wines

Butyltin compounds are widespread contaminants that have also been found in some wines. The purpose of the present work was to make a survey of butyltin compounds in Portuguese wines. Forty-three table wines and 14 Port wines were analyzed for butyltin contents by using solid-phase microextraction gas chromatography mass spectrometry (SPME-GC-MS). In 14% of the analyzed wine samples, measurable dibutyltin (DBT) was found at concentrations ranging between 0.05 and 0.15 microg/L as Sn. Monobutyltin (MBT) was also observed (0.05 microg/L as Sn) in just a single wine. A search for the possible sources of DBT residues found in the wines was carried out. Therefore, some plastics and oak wood used in the process of wine-making, which have been directly in contact with the musts or the wines, were studied to check their possible release of butyltins. The eventual presence of DBT was also tested directly along the vinification process, from the must to the finished product. The results suggest that high-density polyethylene containers used in the transfer of wine in an early stage of the vinification process may be the main sources of these contaminants. Therefore, it is recommendable that plastic materials to be used in wineries be previously tested for the release of butyltin compounds.     

Influence of lysozyme treatments on champagne base wine foaming properties

The objective of this study was to estimate the effect of lysozyme on the foaming characteristics of Champagne base wine. Lysozyme additions were made to the musts and also to the wines before and after bentonite or charcoal treatments, which remove endogenous proteins. Treatments with bentonite diminished foamability and foam stability of wines, whatever the dose (30 or 80 g/hL) and variety [Chardonnay, -28%; Pinot noir, -20% (at 30 g/hL)]. An addition of lysozyme in must raised Pinot noir wine foamability by 21%, whereas the difference is hardly perceptible for Chardonnay wine (+3%). Pinot noir and Chardonnay wines, originating from lysozyme-treated musts, in addition to bentonite treatment on the wine, presented higher foamability than wines treated only with bentonite. Lysozyme was removed (91-100%) by the bentonite treatment. Then, it was not responsible for the increase in foamability but seemed to have a protective effect on the wine proteins. When wines were initially treated with bentonite (150 g/hL) and then enriched with 80 g/hL lysozyme, this enzyme was not able to restore foaming properties. Treatments with charcoal always diminished foamability. The average increase in foamability due to an addition of lysozyme after charcoal treatment (80 g/hL) was 23%. Results showed a real positive effect of lysozyme on foam stability when wines have to be treated with charcoal (+25% and +56% for the Pinot noir wine and the Chardonnay wine, respectively, at 30 g/hL).     

Determination of free proline and monosaccharides in wine samples by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD)

A sensitive and selective analytical method for the simultaneous separation and quantitative determination of proline and free monosaccharides in wine samples by high-performance anion-exchange chromatography coupled with pulsed amperometric detection is described. Under optimized experimental conditions, a complete separation was obtained in less than 30 min, using an isocratic elution with 10 mM NaOH and 1 mM Ba(OAc)(2). No postcolumn addition of strong bases to the eluent for enhancing detection sensitivity was needed. Upon 25-fold sample dilution and purification to avoid interference of tannins, pigments, and phenolic compounds, the fingerprinting of common monosaccharides (i.e., arabinose, glucose, fructose, galactose, and xylose) and proline in wines, musts, and vinegars can be easily accomplished. The method allows high recovery and satisfies the necessary requirements for accuracy, repeatability, and sensitivity. Values obtained for proline content ranged from 470 to 1190 mg/L in "Aglianico" red wines (mean value, 870 +/- 192 mg/L, n = 21) and from 168 to 286 mg/L in white wines (mean value, 208 +/- 32 mg/L, n = 11). Lower levels were found in musts of red and white grapes, 550 and 87 mg/L, respectively. The lowest content of proline, ca. 10 mg/L, was found both in white and red vinegars.     

Method for the gas chromatographic assay with mass selective detection of trichloro compounds in corks and wines applied to elucidate the potential cause of cork taint

To investigate the role of trichloro compounds as a potential cause of "cork taint" in wine, an assay for trichloroanisole (TCA) and trichlorophenol (TCP) in corks and wine was developed utilizing solid phase extraction on a C(18) cartridge followed by gas chromatography with mass selective detection. Recovery and imprecision for TCA were 86-102 and 1.6-5.8%, respectively, and for TCP 82-103% and 1.7-3.9%, respectively. Limits of detection and quantitation were 0.1 and 2 ng/L, respectively, for TCA, and 0.7 and 4 ng/L, respectively for TCP. A survey of 2400 commercial wines revealed a higher incidence of cork taint in white wine than in red and in wines utilizing composite cork closures; wines from central Europe and Spain had higher overall rates of contamination and those from Canada and Italy the lowest. Significant but modest associations were found between the TCA and TCP contents of the wines and corks, but many wines exhibiting cork taint had low or undetectable concentrations of TCA. Over a 12-month period, experimentally bottled wines exhibited a slow increase in TCA and TCP content while cork closures manifested a decrease; most bottles showing cork taint contained low levels of TCA, and TCP concentrations were well below the sensory threshold. Neither compound was cytotoxic to human cell lines in culture up to final concentrations of 500 ng/mL. It was concluded that these two trichloro compounds are, at most, minor components of cork taint in commercial wines.     

Impact odorants of different young white wines from the Canary Islands

Five young monovarietal white wines from the Canary Islands made from Gual, Verdello, Marmajuelo, white Listán, and Malvasia grape cultivars were studied to determine the characteristics of their most important aromas and the differences among them. The study was carried out using gas chromatography-olfactometry (GC-O) to detect the potentially most important aroma compounds, which were then analyzed quantitatively by gas chromatography-flame ionization detection and gas chromatography-mass spectrometry. The strongest odorants in the GC-O experiments were similar in all cases, although significant differences in intensity between samples were noted. Calculation of the odor activity values (OAVs) showed that 3-mercaptohexyl acetate was the most active odorant in the Marmajuelo and Verdello wines, as were 3-methylbutyl acetate in the Gual wine, beta-damascenone in the Malvasia wine, and ethyl octanoate in the white Listán wine. However, the most important differences between varieties were caused by the three mercaptans (3-mercaptohexyl acetate, 3-mercaptohexanol, and 4-methyl-4-mercapto-2-pentanone) and the vinylphenols (4-vinylphenol and 2-methoxy-4-vinylphenol). The correlation between the olfactometric values and the OAVs was satisfactory in the cases when the compound eluted in the GC-O system was well isolated from other odorants and had aromatic importance and the OAVs for the different wines were sufficiently different.     

Polysaccharide profile and content during the vinification and aging of Tempranillo red wines

Passing from must to wine produced a loss of low-molecular-weight grape structural glucosyl polysaccharides, and an important gain in yeast mannoproteins (MP) and grape-derived arabinogalactan proteins (AGP), and rhamnogalacturonans-II (RG-II). AGP were more easily extracted than RG-II, and small quantities of RG-II monomers and galacturonans were detected. Postmaceration produced a reduction in all grape polysaccharide families, particularly acute in AGP. The reduction of polysaccharides during malolactic fermentation only affected grape AGP, and MP were continuously liberated during the entire vinification process. Wine oak and bottle aging was associated with a relative stability of the polysaccharide families. AGP were thus the majority polysaccharides in young wines but, contrary to what may be thought, structural glucosyl oligosaccharides dominated in musts and MP in aged wines. Precipitation of polysaccharides was noticeable during vinification, and it mainly affected high-molecular-weight AGP and MP. Hydrolytic phenomena affected the balance of wine polysaccharides during late maceration-fermentation.     

Determination of stilbenes (trans-astringin, cis- and trans-piceid, and cis- and trans-resveratrol) in Portuguese wines.

Stilbenes have been shown to have cancer chemopreventive activity and to protect lipoproteins from oxidative damage. A method is described for their direct determination in different types of wine using high-performance liquid chromatography with ultraviolet detection. In a survey of 120 commercial wines from Portugal and France, the highest concentrations of stilbenes were found in red wines. The glucosides of resveratrol were present in higher concentrations than the free isomers. Isolation from wine and characterization of trans-astringin in a large quantity are described for the first time.     

Biological activity of total lipids from red and white wine/must.

Wine is an essential component of the Mediterranean diet, and it is thought to exert a protective effect against coronary heart disease. Although many efforts have been made to determine the protective compounds in wines, their exact nature and how they are involved in the protection mechanisms are still unclear. In this study, total lipids, total polar lipids, and total neutral lipids of five wines and three musts were tested in vitro for their ability to induce washed rabbit platelet aggregation and/or to inhibit platelet activating factor (PAF) induced aggregation. The results showed that the biological activity of wine/must total lipids can be attributed mainly to total polar lipids. In the red wine Cabernet Sauvignon, we fractionated total neutral lipids, total polar lipids, and pigments by HPLC. Each fraction was tested in vitro for its biological activity. Structural data of the most active fractions, based on biological, chemical, and spectral methods, are also presented.     

Associations between the sensory attributes and volatile composition of Cabernet Sauvignon wines and the volatile composition of the grapes used for their production

The sensory properties of wine are influenced by the chemical composition of the grapes used to produce them. Identification of grape and wine chemical markers associated with the attributes perceived by the consumer of the wine will enable better prediction of the potential of a parcel of grapes to produce wine of a certain flavor. This study explores the relationships between Cabernet Sauvignon grape volatile composition and wine volatile profiles with the sensory properties of wines. Twenty grape samples were obtained from nine vineyard sites across three vintages and wines vinified from these parcels using controlled winemaking methods. The volatile composition of the grapes were analyzed by SBSE-GCMS, the wines were analyzed by SPME-GCMS, and these data sets were compared to that obtained from the sensory analysis of the wines. Statistical treatment of the data to account for vintage and region effects allowed underlying relationships to be seen between wine sensory attributes and wine or grape volatile components. The observed associations between grape or wine volatile compounds and wine sensory attributes has revealed target compounds and pathways whose levels may reflect the biochemical effects on grape composition by differing growth conditions during berry development and ripening. The compounds identified in this study may be useful grape or wine markers for potential wine sensory characteristics.     

Optimization of making barrel-fermented dry muscatel wines

The optimization of making barrel-fermented muscatel wines requires determining what type of must clarification is most suitable for the quality of the wine, as well as what type of barrel will yield the most acceptable wines. This is achieved by adding pectolytic enzymes to clarify part of the muscatel must statically; the rest is clarified by vacuum filtration. The musts obtained are fermented in French and American oak barrels and, once fermentation has ceased, they are kept with their lees for 2 months, with periodic stirring. Eleven conventional parameters and 31 volatile compounds were quantified, and a sensory analysis of the wines was produced, which led us to conclude that static clarification with pectolytic enzymes from the muscatel musts produces the best-structured wines and the larger content of higher alcohols, esters, and terpenic compounds. The wines fermented in American oak barrels received the highest overall marks, which may be due to the greater aromatic complexity given off by the compounds in the wood.     

Hydroxycinnamic acid ethyl esters as precursors to ethylphenols in wine

A method for determining ethyl coumarate and ethyl ferulate in wine using GC-MS with deuterium-labeled analogues has been developed and used to measure the evolution of these two esters during the production of two commercial monovarietal red wines, cv. Grenache and Shiraz. During fermentation, the concentration of ethyl coumarate rose from low levels to 0.4 mg/L in Grenache and 1.6 mg/L in Shiraz wines. These concentrations then increased further during barrel aging to 1.4 and 3.6 mg/L, respectively. The concentration of ethyl ferulate was much lower, reaching a maximum of only 0.09 mg/L. Conversion of ethyl coumarate and ethyl ferulate to their corresponding ethylphenols was observed during fermentations of a synthetic medium with two strains of Dekkera bruxellensis (AWRI 1499 and AWRI 1608), while a third (strain AWRI 1613) produced no ethylphenols at all from these precursors. Strains AWRI 1499 and 1608 produced 4-ethylphenol from ethyl coumarate in 68% and 57% yields, respectively. The corresponding yields of 4-ethylguaiacol from ethyl ferulate were much lower, 7% and 3%. Monitoring of ethyl coumarate and ethyl ferulate concentration during the Dekkera fermentations showed that the selectivity for ethylphenol production according to yeast strain and the precursor was principally a result of variation in esterase activity. Consequently, ethyl coumarate can be considered to be a significant precursor to 4-ethylphenol in wines affected by these two strains of Brettanomyces/Dekkera yeast, while ethyl ferulate is not an important precursor to 4-ethylguaiacol.     

Determination of 24 pesticide residues in fortified wines by solid-phase microextraction and gas chromatography-tandem mass spectrometry

The present work describes a solid-phase microextraction (SPME) gas chromatography-tandem mass spectrometry (MS/MS) method to quantify 24 pesticides in fortified white wine and fortified red wine. In this study "fortified wine" refers to a wine in which fermentation is arrested before completion by alcohol distillate addition, allowing sugar and alcoholic contents to be higher (around 80-100 g/L total sugars and 19-22% alcohol strength (v/v)). The analytical method showed good linearity, presenting correlation coefficients (R(2)) ≥ 0.989 for all compounds. Limits of detection (LOD) and quantitation (LOQ) in the ranges of 0.05-72.35 and 0.16-219.23 μg/L, respectively, were obtained. LOQs are below the maximum residue levels (MRL) set by European Regulation for grapes. The proposed method was applied to 17 commercial fortified wines. The analyzed pesticides were not detected in the wines tested.     

Development of a rapid "fingerprinting" system for wine authenticity by mid-infrared spectroscopy

This paper reports on the development of a rapid and simple method for red wine authenticity confirmation during transport and processing; namely, a wine "fingerprinting" system. When wine is transported between two sites, a sample is taken and a mid-infrared (MIR) spectrum is obtained. One hundred sixty-one (n = 161) samples of three main red wine varieties grown in Australia, Shiraz, Cabernet Sauvignon, and Merlot, were collected from six commercial wineries across Australia and scanned in transmission on two MIR spectrophotometers located at The Hardy Wine Company's main site at Reynella, South Australia (Foss WineScan FT 120) (926-5012 cm-1). A similarity index (SI) method was used as a tool to classify wine samples on the basis of their spectral data. The results showed that high rates of classification were obtained when wine samples scanned in different instruments were analyzed. The SI has been proven to provide an acceptable measurement for authentication of red wine integrity during transportation. In five of the six winery data sets, the SI correctly classified 98% of the wines. It was also observed that less than 1% of wines were misclassified between the different wineries investigated. Further studies are needed in order to test the applicability of the SI in a commercial situation and to evaluate its potential as a rapid quality control tool for routine use to authenticate wine samples during transport.