Jorge M. Ricardo-da-Silva

CHEMICAL CHARACTERIZATION AND ANTIOXIDANT ACTIVITIES OF OLIGOMERIC AND POLYMERIC PROCYANIDIN FRACTIONS FROM GRAPE SEEDS

Two procyanidin fractions, namely oligomers and polymers isolated from grape seed methanolic extract were characterized. Phenolic composition and procyanidin purity of these fractions were determined by normal-phase and reverse-phase HPLC, thioacidolysis-HPLC, ESI-MS analyses, formaldehyde-HCl precipitation and elemental analysis. Antioxidant activities of these fractions and other well-known antioxidants were measured using xanthine-xanthine oxidase system for generating superoxide radical ({O2(-)}), the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical method and the Fenton system for generating hydroxyl radical (HO). The results showed that both oligomeric and polymeric procyanidin fractions were highly pure, with the degree of polymerization ranging from 2 to 17-18 and 12 to 32-37, respectively. On the basis of molar concentration, polymeric procyanidins appeared the highest antioxidant activities, followed by oligomeric procyanidins, whereas catechins presented a lower antioxidant activity than its oligomers and polymers. These results indicate that the antioxidant activities of grape seed procyanidins are positively related to their degree of polymerization. Moreover, grape seed procyanidins presented higher antioxidant activities than other well-known antioxidants such as vitamin C, suggesting that grape seed procyanidins might be of interest to be used as alternative antioxidants.

Interactions of Oligomeric Procyanidins in Model Wine Solutions Containing Malvidin‐3‐Glucoside and Acetaldehyde

Interactions of grape seed flavanols, dimeric, galloylated dimeric and trimeric procyanidins with malvidin-3-glucoside and acetaldehyde were studied in wine-like model solutions. Malvidin-3-glucoside and oligomeric procyanidins reacted very slowly, with eventual formation of yellow xanthylium salts. The concentrations of malvidin-3-glucoside and oligomeric procyanidins decreased faster in the presence of acetaldehyde and the colour augmentation with shift towards violet was attributed to the formation of new coloured compounds, detectable by HPLC and linked at different positions by CH 3 CH bridges. The rate of loss of procyanidins in the presence of malvidin-3-glucoside and acetaldehyde varied according to the complexity of the components. Monomeric (+)-catechin and (-)-epicatechin reacted more slowly than dimer procyanidins B 1 , B 2 and B 3 while trimer C 1 (epicatechin 4β → 8 epicatechin 4β → 8 epicatechin) presented the highest degradative reaction rate.

Volatile composition analysis by solid-phase microextraction applied to oak wood used in cooperage (Quercus pyrenaica and Quercus petraea) : effect of botanical species and toasting process

The purpose of this study was to investigate the sorption of selected volatile substances from oak wood-chip samples (Quercus pyrenaica Willd. and Quercus petraea L.) subjected to different toasting levels, namely, without toasting, with medium toasting, and with strong toasting, through the use of solid-phase microextraction (SPME). The main volatile compounds identified as a function of the toasting level and botanical species were furfural, hexanal, α-pinene, d-limonene, decanal, vitispirane, ethyl hexanoate, cis-3-methyl-γ-octalactone (“oak lactone” or “whisky lactone”), α-terpineol, p-xylene, and nonanal. Considering the data obtained from the toasted woods (medium and strong intensity) in comparison with those of nontoasted woods, it can be pointed out that the average peak area and the number of compounds identified in the gas chromatogram decreased during the toasting process. In general, regarding the compounds analyzed, quantitative differences were found between the two oak wood species under study. High values of volatile compounds were found in Quercus pyrenaica oak wood chips. In addition, for the number of compounds identified in oak wood extracts and directly extracted from solid oak wood chips by SPME, it is concluded that the best extraction process for volatile compounds from oak wood is the use of oak wood-chip liquid extracts.

Effect of Saccharomyces strains on the quality of red wines aged on lees

Ageing on lees involves ageing the wine in contact with yeast cells after fermentation. If combined with the addition of oak chips, it can soften the wood flavour and increase the aromatic complexity of wine. The aim of the present work is to optimise both ageing techniques through selection of an adequate Saccharomyces cerevisiae strain. The study lasted 6 months and content of polysaccharides, anthocyanins, proanthocyanidins, volatile compounds, colour parameters and sensory analysis, were periodically evaluated. Among the strains tested, G37 showed the highest release of polysaccharides (24.4±5.5 mg l(-1)). Vanillin, syringaldehyde and furfuryl alcohol increased with ageing time in 7VA2 treatment. The wine aged with CTPL14 strain presented fewer monomeric and oligomeric proanthocyanidins (12.4±0.6 and 83.4±8.3 mg l(-1), respectively), and showed the lowest astringency and bitterness sensations. Results show an improvement in the sensory profile of the red wine aged with a combination of these two techniques.

Ellagitannins from Portuguese oak wood (Quercus pyrenaica Willd.) used in cooperage: influence of geographical origin, coarseness of the grain and toasting level

Abstract The ellagitannins and ellagic acid from extracts of Portuguese oak wood chips (Quercus pyrenaica) used in cooperage from different origins (Gerês and Guarda forests) were analysed by HPLC. The results were compared with the ellagitannin content of wood chips obtained from American (Q. alba) and French (Q. petraea) oak. The influence of heat treatment and the coarseness of the grain on ellagitannins and ellagic acid were also studied. Five ellagitannins (vescalagin, castalagin, roburin D, roburin E and grandinin) and ellagic acid were quantified. Vescalagin and castalagin were the most abundant individual ellagitannins in all of the oak wood chips studied. Portuguese oak wood had the highest content of ellagitannins and ellagic acid, followed by the French and American oaks. The geographical origin of Portuguese oak and the coarseness of the grain of French oak affected the content of ellagitannins and ellagic acid. In general, the ellagitannin content decreased with the toasting process, while the ellagic acid content increased. The toasting process had the highest impact on ellagitannin composition in comparison to the other variables studied.

Vitis vinifera secondary metabolism as affected by sulfate depletion: diagnosis through phenylpropanoid pathway genes and metabolites

Grapevine (Vitis vinifera L.) is rich in phenylpropanoid compounds, namely flavonoids and stilbenes which, present in most tissues, are described as antioxidants and known to accumulate in response to biotic and abiotic stress. Grapevine is then a choice model for studying the interplay between the phenylpropanoid pathway and nutrient deficiency. Here we report the response to sulfur deficiency (-S) of flavonoids and stilbenes biosynthetic pathways in chlorophyll tissues (plantlets) and cell culture. Anthocyanins and trans-resveratrol accumulated in plantlets and trans-resveratrol glucoside in cell cultures in response to sulfur deficiency, while a significant decrease in chlorophyll was observed in -S plantlets. The up-regulation of chalcone synthase gene and the downstream flavonoid biosynthesis genes dihydroflavonol reductase and anthocyanidin synthase matched the accumulation of anthocyanins in -S V. vinifera plantlets. The mRNA level of stilbene synthase gene(s) was correlated tightly with the increase in trans-resveratrol and trans-resveratrol glucoside levels, respectively in -S plantlets and cell cultures. As a whole, the present study unveil that V. vinifera under sulfur deficiency allocates resources to the phenylpropanoid pathway, probably consecutive to inhibition of protein synthesis, which can be advantageous to resist against oxidative stress symptoms evoked by -S conditions.

Effect of Winery Yeast Lees on Touriga Nacional Red Wine Color and Tannin Evolution

Red wine aging on lees is a winemaking practice used to achieve more rounded and less astringent wines. In two different trials, external yeast lees were added to a red wine and their effects on wine color and tannin evolution during aging were studied. Results indicated that the addition of yeast lees did not affect color stabilization during the studied period. Color compounds and condensed tannins were rapidly adsorbed to the yeast lees at the beginning of the experiment. There was a retarding effect on proanthocyanidin polymerization reaction by the addition of yeast lees, leading to the maintenance of low and medium molecular weight tannins in solution. Two different interactions were observed: first, proanthocyanidin adsorption by the yeast lees, primarily ones with the highest polymerization degree, and second, the retarding of proanthocyanidin polymerization, likely by the mannoproteins released by yeast lees. The age of the yeast lees was a factor in mannoprotein release and its effect in wine.

Extraction of Some Ellagic Tannins and Ellagic Acid from Oak: Wood Chips (Quercus pyrenaica L.) in Model Wine Solutions: Effect of Time, pH, Temperature and Alcoholic Content

The subject of the present work is the study of the influence of pH, temperature, alcoholic content and time on the extraction of some individual ellagic tannins (castalagin, vescalagin, grandinin, roburin D and E) and ellagic acid from oak wood chips (Quercus pyrenaica L.) within model wine solutions. The determination of these compounds by reversed-phase high-performance liquid chromatography (HPLC), after 104 extraction days, enabled us to establish the qualitative and quantitative evolution of each component and the effect of each extraction condition on individual and total ellagic tannins from oak wood chips. Vescalagin and castalagin were the most abundant individual ellagic tannins measured under all extraction conditions. Individual ellagic tannins and ellagic acid increased during the first weeks of extraction, followed by a decrease. Under the extraction conditions examined, temperature was the main factor influencing ellagic tannins and elagic acid evolution. The results suggest that a decrease/degradation of these compounds is less noticeable at low temperatures (12oC). After 104 extraction days the ellagic tannins content in a model wine solution at 12oC was higher than the content of ellagic tannins in solutions at 20oC. On the other hand, the effects of alcoholic content and pH of model wine solutions on the extraction and evolution of the analysed compounds (except for castalagin and vescalagin) seemed to be less important than temperature.

Kinetics of odorant compounds in wine brandies aged in different systems.

The odorants compounds of aged wine brandies comprise compounds deriving from the wood, from the distillate and from the reactions that occur inside the barrel. The aim of this work was to study the kinetics of the odorant compounds of a wine brandy during two years of ageing in two ageing systems. The odorant compounds in the analysed brandies changed significantly over the time, but with different evolution patterns. The wood related compounds increased over time, with the highest increase in the first months of ageing. The kinetics of cis, trans-β-methyl-γ-octalactone, acetovanillone and of seven volatile phenols are established for the first time in brandies. Moreover, a significant effect of the ageing system was found on the kinetics of the wood related compounds. These results pointed out the interest of these compounds as a tool to discriminate different ageing technologies.

Evaluation of Portuguese and Spanish Quercus pyrenaica and Castanea sativa species used in cooperage as natural source of phenolic compounds

A study on the phenolic composition of Portuguese and Spanish Castanea sativa and Quercus pyrenaica woods used in cooperage has been carried out with the aim of looking for an extra value for the wood residues generated in cooperage industries. Results showed that both species are an important source of valuable phenolic compounds, although differences in the phenolic content were found. Chestnut samples showed the highest concentration of total phenolic compounds, being especially rich in vescalagin, gallic acid, vanillic aldehyde and syringaldehyde. The geographical origin was another parameter to take into account. Portuguese chestnut samples showed higher amounts of phenolic compounds than the Spanish samples, with vescalagin, gallic acid, vanillic acid and syringaldehyde being the most important features. As a consequence, the residues generated in the cooperage industry from chestnut and Q. pyrenaica heartwood seem to be an important source of valuable phenolic compounds, particularly those from Portuguese chestnuts.