These studies explored interactions between flavanols and nitric oxide in order to investigate implications for vascular health. Study 1 investigated acute effects of flavanol consumption on cutaneous microvascular endothelial function in young and older individuals along with chronic exposure in older individuals. This was accomplished by assessing skin blood flow response to local heating (thermal reactivity, TR); skin was clamped at 34°C and 40°C and values were normalized to those attained at 43°C. Older individuals demonstrated an attenuated TR at baseline during the entire local heating phase (58.4 ± 2.5% versus 49.3 ± 2.6%, p<0.05). Acutely following flavanol ingestion there was a significant increase in TR (52.4 ± 2.1% versus 56.1 ± 2.0%, p=0.05) that was not different with age. There was no effect of chronic flavanol exposure on TR in older individuals; however, there was a significant decrease in mean arterial pressure (95 ± 3 mmHg versus 91 ± 3 mmHg, p<0.001). These results contribute to research regarding flavanols increasing NO bioavailability; acutely via an improvement in cutaneous microvascular endothelial function and chronically via a reduction in blood pressure. Study 2 investigated the acute effects of flavanol consumption on cerebrovascular endothelial function in young and older individuals along with chronic flavanol exposure in older individuals. This was accomplished by assessing basal cerebral blood flow indices (cerebral vascular conductance index, CVCi) and CBF response to hypercapnia (cerebral vasomotor reactivity; CVMR). At baseline older individuals demonstrated a reduced CVCi (0.85 ± 0.04 cm/s*mmHg versus 0.55 ± 0.04 cm/s*mmHg p=0.001) and CVMR (8.6 ± 0.6 versus 6.9 ± 0.4, p=0.05). An unexpected finding was that flavanol ingestion led to an acute decrease in CVCi (0.71 ± 0.04 cm/s*mmHg versus 0.62 ± 0.04 cm/s*mmHg p<0.05) and CVMR (8.6 ± 0.6 versus 6.1 ± 0.5, p=0.001) that was not different with age. In older individuals, chronic exposure led to a significant increase in CVCi (0.60 ± 0.05 cm/s*mmHg versus 0.72 ± 0.06 cm/s*mmHg, p<0.05) but had no effect on CVMR. These data provide evidence for an improvement in cerebral hemodynamics following chronic exposure in older individuals. / text

Le vin est un milieu complexe qui évolue tout au long des étapes devinification. Pour mieux appréhender ses qualités et ses défauts, de nombreuses équipesde recherche s’intéressent à la compréhension de la chimie du vin. Dans ce contexte, lesujet de ce mémoire concerne l’étude de l’évolution chimique de certaines moléculespolyphénoliques du vin sous conditions oxydantes et/ou acides, afin d’isoler et decaractériser de nouveaux composés susceptibles de se former dans le vin. Deux aspectssont examinés. Le premier concerne l’oxydation de deux flavano‐ellagitannins, lesacutissimines A et B, formées à partir d’un monomère de tannins condensés, lacatéchine, et d’un ellagitannin C‐glucosidique, la vescalagine, extraite du bois de chênepar le vin lors de l’élevage en barrique. Cette étude a permis d’isoler les mongolicaïnes Aet B et deux analogues du camelliatannin G et de mettre en évidence leur formation parun mécanisme d’autoxydation. Le deuxième aspect concerne l’évaluation desconséquences de la présence de certains polyphénols dans le vin sur les composésthiolés odorants. Leur comportement et leur réactivité chimiques sont décrits dans desmilieux différents, avec l’hémisynthèse de thio‐ellagitannins sous conditions acides, et laformation d’adduits thio‐catéchols et thio‐pyrogallols sous conditions oxydantes,transformations chimiques pouvant occasionner la perte des odeurs et arômes du vindus aux composés thiolés odorants. / The wine is a complex medium that evolves throughout the different stages ofthe wine making process. To understand both the qualities and defects of wine,numerous research team worldwide investigate the chemistry of wine. In this context,the subject of this thesis concerns the study of the chemical evolution of some winepolyphenolic molecules under oxidizing and/or acidic conditions in the aim of isolatingand characterizing new compounds likely formed in wine. Two aspects are examined.The first one is the study of the oxidation of two flavano‐ellagitannins, acutissimins Aand B, formed from a monomer of condensed tannins, catechin, and a C‐glucosidicellagitannin, vescalagin, extracted from oak wood by the wine solution during its agingin barrels. This study led to the isolation of mongolicains A and B and two analogues ofcamelliatannin G, and revealed their formation according to an autoxydationmechanism. The second aspect of this work concerns the consequences of the presencein wine of some polyphenols on wine odorous thiols. Their chemical behavior andreactivity are described in different media, with the hemisynthesis of thio‐ellagitanninsunder acidic conditions, and the formation of thio‐catechol and thio‐pyrogallol adductsunder oxidizing conditions, chemical transformations that could explain the loss ofodors and aromas due to wine odorous thiols.

Ce travaille de thèse concerne la synthèse de dérivés de polyphénols de type flavanol, ellagitannins C-glucosidique et procyanidine, modifiés avec un espaceur comportant une biotine terminale. Cette biotine terminale a permis d’immobiliser ces polyphénols modifiés sur des surfaces SPR, permettant ainsi l’étude d’interactions polyphénol-protéine en temps réel. Ainsi, la topoisomerase II alpha et l'actine fibrilaire ont montré une plus grande affinité pour les polyphenols de type ellagitannins que pour ceux de type flavanol. Nous avons également pu montrer que d’autres protéines (BSA, myoglobine, actine globulaire, streptavidine, collagen type I) n’avaient pas d’interaction avec les flavanols et les ellagitannins. / This work concerns the synthesis of derivatives of polyphenols of the type flavanol, C-glucosidic ellagitannin and procyanidin, which are modified to bear a spacer ending with a biotin unit. This biotin ending unit allowed to immobilize these modified polyphenols on to SPR surfaces, which allowed the study of polyphenol-protein interactions in real time. The proteins topoisomerase II alpha and fibrilar actin showed a higher affinity for the polyphenols of the type ellagitannins than for those of the type flavanol. It was also showed that other proteins (BSA, myoglobin, globular actin, streptavidin, collagen type I) did not interact with either the flavanols or the ellagitannins

Dans le vin rouge, les composés phénoliques sont souvent associés à la qualité du produit. Parmi eux, les anthocyanes sont responsables de la couleur du vin rouge et chaque cépage possède un génome spécifique déterminant le profil anthocyanique de chacun. Les pratiques culturales peuvent influencer l’accumulation de ces molécules dans la baie de raisin, tandis que les différentes techniques fermentaires peuvent modifier leur extraction. Les flavanols monomères, oligomères et polymères des pépins et des pellicules contribuent à l’amertume et l’astringence des vins. Pendant la vinification et l’élevage, ceux-ci réagissent avec les anthocyanes modifiant les propriétés du vin. De nombreuses publications concernant les caractéristiques sensorielles des flavanols ont été réalisées, alors que la contribution sensorielle des anthocyanes est encore mal connue. Cette thèse se décompose en deux parties. La première partie s’intéresse à l’étude de l’utilisation d’ozone gazeux comme technique pré-fermentaire sur les cépages rouges Nebbiolo et Barbera après récolte et pendant le passerillage. L’ozone est une technique innovante pour la réduction des contaminations microbiennes et la réduction du dioxyde de soufre. L’influence de cette technique sur les parois cellulaire des pellicules, sur l’extraction, la concentration en flavanols et en anthocyanes pendant la macération a été évaluée. L’étude montre que l’ozone possède un impact sur la macération des raisins, induisant une extraction plus importante des anthocyanes dans le Nebbiolo. En revanche, l’ozone n’influence ni les anthocyanes moléculaires, ni le profil variétal de chaque cépage. Pendant le passerillage, l’inverse a été étudié. L’extraction des anthocyanes dans le Nebbiolo est diminuée. A l’inverse, bien que moins d’anthocyanes soient retrouvées dans le raisin de Barbera, le traitement ne possède aucun impact sur l’extraction de celles-ci. Concernant les flavanols, l’extraction est moins influencée par le traitement à l’ozone. Des différences ont été retrouvées dans le cépage Nebbiolo. Les flavonols sont plus concentrés après le traitement avec l’ozone sur les raisins post-récolte, alors qu’ils diminuent pendant le passerillage. La modification causée sur la paroi cellulaire par l’ozone ainsi que la dureté des pellicules pourraient prédire l’extraction des anthocyanes et des flavanols, grâce à des analyses multi-variées. En conséquence, le traitement avec l’ozone doit être adapté en fonction du cépage et du produit final désiré. Dans la deuxième partie, les anthocyanes des raisins ont été extraites à partir des raisins et ces extraits ont été fractionnés en trois fractions, glucoside, acetyl-glucoside et cinnamoyl-glucoside, par Chromatographie de Partage Centrifuge (CPC) et CLHP (Chromatographie Liquide Haute Performance) préparative. L’évaluation de l’astringence a été réalisée par des analyses de précipitation protéique avec une protéine modèle BSA (Bovine Serum Albumin) et des protéines salivaires, les anthocyanes réagissant avec ces dernières. Les analyses sensorielles et l’évaluation de l’astringence ont été combinées. La concentration en anthocyane diminue après le traitement avec les protéines salivaires, dans l’extrait total et dans les fractions, en particulier les cinnamoyl-glucosides apparaissent comme les plus réactives avec les protéines salivaires. Les seuils de perception gustatifs ont été calculés avec la méthode “Best estimate threshold” dans le vin modèle. Les fractions acétyl-glucosides et cinnamoyl-glucosides, suivies de la fraction glucoside, possèdent des seuils de perception plus bas aux concentrations retrouvées dans les vins. Les descripteurs associés à ces fractions sont l’amertume et l’astringence. Ces résultats démontreraient que les anthocyanes apportent une contribution sensorielle dans la perception du vin en bouche, corrélée à l’acétylation des molécules. / In red wine, phenolic compounds are generally associated with the quality of products. Among them, anthocyanins extracted from skins are responsible for wine colour. The grapevine genomes determine the anthocyanins profiles, but several factors in the vineyard can influence their accumulation, as well as post-harvest techniques can modify their extraction during winemaking. Monomeric, oligomeric and polymeric flavanols from skins and seeds contribute to astringency and bitterness of wine and during winemaking and ageing complexes formation with anthocyanins modifies wine characteristics. Several publications are available to understand flavanols sensory characteristics, whereas anthocyanins role has not consensus in scientific literature. This PhD thesis is composed by two parts. The first part deal with the evaluation of the use of gaseous ozone as post-harvest technique in red wine grapes Nebbiolo and Barbera used on both fresh grape and during withering. Ozone treatment is an innovative technology proved to avoid mycobiota spoilage and preserving from the use of sulphur dioxide. Its influence on flavanol and anthocyanin contents and extractabilities during maceration was evaluated, considering skin cell wall modification. In fresh grape, ozone influenced skin maceration for both the varieties, leading to a higher anthocyanin extraction in Nebbiolo grapes and lower in Barbera. Ozone did not influence the final individual anthocyanin extractability, respecting the varietal anthocyanin fingerprint. During dehydration, opposite trend was found: in Nebbiolo reported no change in the content of total anthocyanins just after ozone-assisted dehydration, but their extraction yield was lower. On the contrary, although lower contents of anthocyanins were found in Barbera grapes no differences in final extractability was found. Regarding oligomeric and polymeric flavanols, their extractability was less affected by the ozone treatment. Only in Nebbiolo, both oligomeric and polymeric flavanol extraction was increased in fresh grape, whereas it is slightly decrease during dehydration. The ozone-induced modification of skin cell wall composition together with skin hardness parameters fitted well in multivariate models to predict anthocyanins, oligomeric flavanols and polymeric flavanols extraction. Therefore, the ozone treatment should be adapted depending on the variety and on the target wine. In the second part, grape anthocyanins were isolated depending on the acylation patterns, i.e. glucoside, acetyl-glucoside, and cinnamoyl-glucoside by a combination of centrifugal partition chromatography (CPC) and preparative-HPLC. Protein precipitation analyses to assess astringency and sensorial analysis were carried out. Anthocyanins reacted with both bovine serum albumine and salivary proteins, in different extent, since higher interaction between anthocyanins and salivary proteins was found with a significative reduction of total extract and fractions glucoside, acetyl-glucoside, and cinnamoyl-glucoside. The latter in particular is the more reactive to salivary proteins. Sensorial analysis was carried out as detection threshold test. Best estimated threshold (BET) of anthocyanins were resulted in wine-range scale, in particular acetyl-glucoside and cinnamoyl-glucoside BET are lower of glucoside threshold, and descriptors reported were astringency and bitterness. These results show that anthocyanins can be detected as in-mouth properties contributors, and the magnitude of their involvement is related to anthocyanins acylation.

O trabalho foi conduzido com uvas Cabernet Sauvignon do distrito de Pinto Bandeira, Bento Gonçalves, RS. Essa cultivar se destaca no Brasil pela produção de vinhos tintos de guarda, tendo-se adaptado relativamente bem na Serra Gaúcha. Como outras cultivares tintas, possue, em determinadas safras, dificuldades para obtenção de uma adequada maturação, dificultando a elaboração de vinhos com boa estrutura. Dentre as estratégias adotadas para minimizar esse problema, está a adoção de um adequado manejo do vinhedo e acompanhamento da maturação, aliados a operações enológicas, como controles na maceração e uso de insumos enológicos. Em função desse contexto objetivou-se estudar a aplicação de taninos enológicos e goma arábica para avaliar este uso na composição físico-química e qualidade sensorial do vinho Cabernet Sauvignon. Na safra 2004 foram usados taninos de quebracho e de castanheira, aplicados em três dosagens (5,0 g.hl-1 , 10,0 g.hL-1, 20,0 g.hl-1 ), e em três momentos de aplicação (maceração, 02 dias após o esmagamento; descuba, 08 dias após o esmagamento; após a fermentação malolática, 04 meses após o esmagamento). Na safra 2005 aplicaram-se os taninos de quebracho, de castanheira e de acácia negra, todos na dosagem de 10,0 g.hl-1 , aplicados na maceração. Ainda nessa safra, os vinhos foram tratados com quatro dosagens de goma arábica (0,0 g.L-1 , 1 ,O g.L-1 , 2,0 g.L-1 e 3,0 g.L-1 ) no ato do engarrafamento. Na safra 2006 aplicaram-se os três taninos testados na safra 2005, todos na dosagem de 20,0 g.hl-1 , durante a maceração, e mais um "pool" de quatro taninos (casca de uva, semente de uva, quebracho, carvalho), na dosagem de 10,0 IPT (Índice de Polifenóis Totais), durante a maceração e no vinho estabilizado. Ainda se testaram duas dosagens de goma arábica (0,0 g.L-1 e 3,0 g.L-1 ), aplicadas no momento do engarrafamento. Nos vinhos estabilizados, realizaram-se as análises físico-químicas clássicas, mais características cromáticas e polifenóis, além de uma análise sensorial detalhada. Em função das boas condições meteorológicas apresentadas nas safras estudadas, que levaram à obtenção de uvas de boa qualidade, no que conceme às características físico-químicas clássicas, de modo geral, se verificou que a adição dos taninos teve pouca influência na composição do vinho. Aquelas variáveis que seriam, hipoteticamente, mais influenciáveis, como antocianinas e índices de cor, mostraram, da mesma forma, poucas mudanças significativas. A goma arábica influenciou uma série de características, denotando que sua utilização afeta de maneira mais significativa a composição do vinho. Na análise sensorial, principalmente o perfil aromático dos vinhos foi influenciado pelo uso dos taninos enológicos, especialmente quando aplicados na maceração. Assim, parece ser prudente inferir que o uso associado de taninos de origens diversas, contribuiu de maneira mais efetiva para a qualidade final dos vinhos. A goma arábica, por sua vez, manteve a tendência de influir positivamente na qualidade gustativa dos vinhos, em especial no aumento da maciez e redução da adstringência. Mesmo que parcialmente, em função das diferentes safras, comprovou-se a expectativa que os taninos enológicos contribuem para aumentar a capacidade antioxidante dos vinhos. / The study was performed with Cabemet Sauvignon grapes from the Pinto Bandeira district, Bento Gonçalves, Rio Grande do Sul, Brazil, a cultivar known throughout the country for the production of cellaring red wine which adapted fairly well to the Serra Gaucha region. Like other red wine vines, there can be obstacles to proper maturation in certain harvests, which can curtail the production of well-structured wines. Among the strategies adopted to minimize the problem are using correct vineyard management and monitoring the maturation process, as well as enological operations such as contrais in maceration and the use of enological inputs. This situation has led to the study of the application of enological tannins and gum arabic to assess their use in the physicochemical composition and sensory properties of Cabernet Sauvignon wine. Quebracho and chestnut tannins were used in the 2004 harvest in three different dosages (5.0 g.hL-1, 10.0 g.hL-1, 20.0 g.hl-1 ) and at three different moments (maceration, 02 days after crushing; devatting, 08 days after crushing; after malolactic fermentation, 04 months after crushing). Quebracho, chestnut and black green-wattle acacia tannins were applied to the 2005 harvest, at 10.0 g.hL-1 dosages, ali during maceration . Wines from the same vintage were also treated with four dosages of gum arabic (0.0 g.L-1 , 1.0 g.L-1 , 2.0 g.L-1 and 3.0 g.L-1 ) at bottling. Three tannins tested on the 2005 harvest were applied to the 2006 one, ali at 20.0 g.hl-1 dosages during maceration, as well as a pool of four tannins (grape skins, grape pips, quebracho, oak) at dosages of 10.0 TPI (Total Polyphenollndex) during maceration and to the stabilized wine. Two gum arabic dosages were also tested (0.0 g.L-1 and 3.0 g.L-1 ), applied at bottling. The classical physicochemical analyses were performed on stabilized wines as well as chromatic characteristics, polyphenols and detailed sensory analysis. Due to the good weather conditions of the vintages studied, which led to the harvest of quality grapes regarding the classical physicochemical characteristics, tannin addition had little overall influence on wine composition. Those variables that would, hypothetically, be the most affected by their addition, such as anthocyanines and colar indexes, did not have significant changes either. Gum arabic influenced several characteristics, which shows that their use has more significant effects on wine composition. In sensory analysis, enological tannins primarily affected the aromatic profile of the wines, especially when applied in the maceration, so it seems safe to inter that the concomitant use of tannins from various origins contributed effectively to the final quality of the wines. Gum arabic, in turn, continued to have a positive effect on the palate of the wines, especially by increasing their softness and decreasing their astringency. The hypothesis that enological tannins contribute to the increase in antioxidant capacity in wines proved correct, even if only partially so due to the different harvests.

O trabalho foi conduzido com uvas Cabernet Sauvignon do distrito de Pinto Bandeira, Bento Gonçalves, RS. Essa cultivar se destaca no Brasil pela produção de vinhos tintos de guarda, tendo-se adaptado relativamente bem na Serra Gaúcha. Como outras cultivares tintas, possue, em determinadas safras, dificuldades para obtenção de uma adequada maturação, dificultando a elaboração de vinhos com boa estrutura. Dentre as estratégias adotadas para minimizar esse problema, está a adoção de um adequado manejo do vinhedo e acompanhamento da maturação, aliados a operações enológicas, como controles na maceração e uso de insumos enológicos. Em função desse contexto objetivou-se estudar a aplicação de taninos enológicos e goma arábica para avaliar este uso na composição físico-química e qualidade sensorial do vinho Cabernet Sauvignon. Na safra 2004 foram usados taninos de quebracho e de castanheira, aplicados em três dosagens (5,0 g.hl-1 , 10,0 g.hL-1, 20,0 g.hl-1 ), e em três momentos de aplicação (maceração, 02 dias após o esmagamento; descuba, 08 dias após o esmagamento; após a fermentação malolática, 04 meses após o esmagamento). Na safra 2005 aplicaram-se os taninos de quebracho, de castanheira e de acácia negra, todos na dosagem de 10,0 g.hl-1 , aplicados na maceração. Ainda nessa safra, os vinhos foram tratados com quatro dosagens de goma arábica (0,0 g.L-1 , 1 ,O g.L-1 , 2,0 g.L-1 e 3,0 g.L-1 ) no ato do engarrafamento. Na safra 2006 aplicaram-se os três taninos testados na safra 2005, todos na dosagem de 20,0 g.hl-1 , durante a maceração, e mais um "pool" de quatro taninos (casca de uva, semente de uva, quebracho, carvalho), na dosagem de 10,0 IPT (Índice de Polifenóis Totais), durante a maceração e no vinho estabilizado. Ainda se testaram duas dosagens de goma arábica (0,0 g.L-1 e 3,0 g.L-1 ), aplicadas no momento do engarrafamento. Nos vinhos estabilizados, realizaram-se as análises físico-químicas clássicas, mais características cromáticas e polifenóis, além de uma análise sensorial detalhada. Em função das boas condições meteorológicas apresentadas nas safras estudadas, que levaram à obtenção de uvas de boa qualidade, no que conceme às características físico-químicas clássicas, de modo geral, se verificou que a adição dos taninos teve pouca influência na composição do vinho. Aquelas variáveis que seriam, hipoteticamente, mais influenciáveis, como antocianinas e índices de cor, mostraram, da mesma forma, poucas mudanças significativas. A goma arábica influenciou uma série de características, denotando que sua utilização afeta de maneira mais significativa a composição do vinho. Na análise sensorial, principalmente o perfil aromático dos vinhos foi influenciado pelo uso dos taninos enológicos, especialmente quando aplicados na maceração. Assim, parece ser prudente inferir que o uso associado de taninos de origens diversas, contribuiu de maneira mais efetiva para a qualidade final dos vinhos. A goma arábica, por sua vez, manteve a tendência de influir positivamente na qualidade gustativa dos vinhos, em especial no aumento da maciez e redução da adstringência. Mesmo que parcialmente, em função das diferentes safras, comprovou-se a expectativa que os taninos enológicos contribuem para aumentar a capacidade antioxidante dos vinhos. / The study was performed with Cabemet Sauvignon grapes from the Pinto Bandeira district, Bento Gonçalves, Rio Grande do Sul, Brazil, a cultivar known throughout the country for the production of cellaring red wine which adapted fairly well to the Serra Gaucha region. Like other red wine vines, there can be obstacles to proper maturation in certain harvests, which can curtail the production of well-structured wines. Among the strategies adopted to minimize the problem are using correct vineyard management and monitoring the maturation process, as well as enological operations such as contrais in maceration and the use of enological inputs. This situation has led to the study of the application of enological tannins and gum arabic to assess their use in the physicochemical composition and sensory properties of Cabernet Sauvignon wine. Quebracho and chestnut tannins were used in the 2004 harvest in three different dosages (5.0 g.hL-1, 10.0 g.hL-1, 20.0 g.hl-1 ) and at three different moments (maceration, 02 days after crushing; devatting, 08 days after crushing; after malolactic fermentation, 04 months after crushing). Quebracho, chestnut and black green-wattle acacia tannins were applied to the 2005 harvest, at 10.0 g.hL-1 dosages, ali during maceration . Wines from the same vintage were also treated with four dosages of gum arabic (0.0 g.L-1 , 1.0 g.L-1 , 2.0 g.L-1 and 3.0 g.L-1 ) at bottling. Three tannins tested on the 2005 harvest were applied to the 2006 one, ali at 20.0 g.hl-1 dosages during maceration, as well as a pool of four tannins (grape skins, grape pips, quebracho, oak) at dosages of 10.0 TPI (Total Polyphenollndex) during maceration and to the stabilized wine. Two gum arabic dosages were also tested (0.0 g.L-1 and 3.0 g.L-1 ), applied at bottling. The classical physicochemical analyses were performed on stabilized wines as well as chromatic characteristics, polyphenols and detailed sensory analysis. Due to the good weather conditions of the vintages studied, which led to the harvest of quality grapes regarding the classical physicochemical characteristics, tannin addition had little overall influence on wine composition. Those variables that would, hypothetically, be the most affected by their addition, such as anthocyanines and colar indexes, did not have significant changes either. Gum arabic influenced several characteristics, which shows that their use has more significant effects on wine composition. In sensory analysis, enological tannins primarily affected the aromatic profile of the wines, especially when applied in the maceration, so it seems safe to inter that the concomitant use of tannins from various origins contributed effectively to the final quality of the wines. Gum arabic, in turn, continued to have a positive effect on the palate of the wines, especially by increasing their softness and decreasing their astringency. The hypothesis that enological tannins contribute to the increase in antioxidant capacity in wines proved correct, even if only partially so due to the different harvests.

O trabalho foi conduzido com uvas Cabernet Sauvignon do distrito de Pinto Bandeira, Bento Gonçalves, RS. Essa cultivar se destaca no Brasil pela produção de vinhos tintos de guarda, tendo-se adaptado relativamente bem na Serra Gaúcha. Como outras cultivares tintas, possue, em determinadas safras, dificuldades para obtenção de uma adequada maturação, dificultando a elaboração de vinhos com boa estrutura. Dentre as estratégias adotadas para minimizar esse problema, está a adoção de um adequado manejo do vinhedo e acompanhamento da maturação, aliados a operações enológicas, como controles na maceração e uso de insumos enológicos. Em função desse contexto objetivou-se estudar a aplicação de taninos enológicos e goma arábica para avaliar este uso na composição físico-química e qualidade sensorial do vinho Cabernet Sauvignon. Na safra 2004 foram usados taninos de quebracho e de castanheira, aplicados em três dosagens (5,0 g.hl-1 , 10,0 g.hL-1, 20,0 g.hl-1 ), e em três momentos de aplicação (maceração, 02 dias após o esmagamento; descuba, 08 dias após o esmagamento; após a fermentação malolática, 04 meses após o esmagamento). Na safra 2005 aplicaram-se os taninos de quebracho, de castanheira e de acácia negra, todos na dosagem de 10,0 g.hl-1 , aplicados na maceração. Ainda nessa safra, os vinhos foram tratados com quatro dosagens de goma arábica (0,0 g.L-1 , 1 ,O g.L-1 , 2,0 g.L-1 e 3,0 g.L-1 ) no ato do engarrafamento. Na safra 2006 aplicaram-se os três taninos testados na safra 2005, todos na dosagem de 20,0 g.hl-1 , durante a maceração, e mais um "pool" de quatro taninos (casca de uva, semente de uva, quebracho, carvalho), na dosagem de 10,0 IPT (Índice de Polifenóis Totais), durante a maceração e no vinho estabilizado. Ainda se testaram duas dosagens de goma arábica (0,0 g.L-1 e 3,0 g.L-1 ), aplicadas no momento do engarrafamento. Nos vinhos estabilizados, realizaram-se as análises físico-químicas clássicas, mais características cromáticas e polifenóis, além de uma análise sensorial detalhada. Em função das boas condições meteorológicas apresentadas nas safras estudadas, que levaram à obtenção de uvas de boa qualidade, no que conceme às características físico-químicas clássicas, de modo geral, se verificou que a adição dos taninos teve pouca influência na composição do vinho. Aquelas variáveis que seriam, hipoteticamente, mais influenciáveis, como antocianinas e índices de cor, mostraram, da mesma forma, poucas mudanças significativas. A goma arábica influenciou uma série de características, denotando que sua utilização afeta de maneira mais significativa a composição do vinho. Na análise sensorial, principalmente o perfil aromático dos vinhos foi influenciado pelo uso dos taninos enológicos, especialmente quando aplicados na maceração. Assim, parece ser prudente inferir que o uso associado de taninos de origens diversas, contribuiu de maneira mais efetiva para a qualidade final dos vinhos. A goma arábica, por sua vez, manteve a tendência de influir positivamente na qualidade gustativa dos vinhos, em especial no aumento da maciez e redução da adstringência. Mesmo que parcialmente, em função das diferentes safras, comprovou-se a expectativa que os taninos enológicos contribuem para aumentar a capacidade antioxidante dos vinhos. / The study was performed with Cabemet Sauvignon grapes from the Pinto Bandeira district, Bento Gonçalves, Rio Grande do Sul, Brazil, a cultivar known throughout the country for the production of cellaring red wine which adapted fairly well to the Serra Gaucha region. Like other red wine vines, there can be obstacles to proper maturation in certain harvests, which can curtail the production of well-structured wines. Among the strategies adopted to minimize the problem are using correct vineyard management and monitoring the maturation process, as well as enological operations such as contrais in maceration and the use of enological inputs. This situation has led to the study of the application of enological tannins and gum arabic to assess their use in the physicochemical composition and sensory properties of Cabernet Sauvignon wine. Quebracho and chestnut tannins were used in the 2004 harvest in three different dosages (5.0 g.hL-1, 10.0 g.hL-1, 20.0 g.hl-1 ) and at three different moments (maceration, 02 days after crushing; devatting, 08 days after crushing; after malolactic fermentation, 04 months after crushing). Quebracho, chestnut and black green-wattle acacia tannins were applied to the 2005 harvest, at 10.0 g.hL-1 dosages, ali during maceration . Wines from the same vintage were also treated with four dosages of gum arabic (0.0 g.L-1 , 1.0 g.L-1 , 2.0 g.L-1 and 3.0 g.L-1 ) at bottling. Three tannins tested on the 2005 harvest were applied to the 2006 one, ali at 20.0 g.hl-1 dosages during maceration, as well as a pool of four tannins (grape skins, grape pips, quebracho, oak) at dosages of 10.0 TPI (Total Polyphenollndex) during maceration and to the stabilized wine. Two gum arabic dosages were also tested (0.0 g.L-1 and 3.0 g.L-1 ), applied at bottling. The classical physicochemical analyses were performed on stabilized wines as well as chromatic characteristics, polyphenols and detailed sensory analysis. Due to the good weather conditions of the vintages studied, which led to the harvest of quality grapes regarding the classical physicochemical characteristics, tannin addition had little overall influence on wine composition. Those variables that would, hypothetically, be the most affected by their addition, such as anthocyanines and colar indexes, did not have significant changes either. Gum arabic influenced several characteristics, which shows that their use has more significant effects on wine composition. In sensory analysis, enological tannins primarily affected the aromatic profile of the wines, especially when applied in the maceration, so it seems safe to inter that the concomitant use of tannins from various origins contributed effectively to the final quality of the wines. Gum arabic, in turn, continued to have a positive effect on the palate of the wines, especially by increasing their softness and decreasing their astringency. The hypothesis that enological tannins contribute to the increase in antioxidant capacity in wines proved correct, even if only partially so due to the different harvests.

Se han obtenido una serie de conjugados de flavanoles a partir de un extracto vegetal mediante un proceso de despolimerización ácida en presencia de derivados de cisteína. Los monómeros de flavanoles derivatizados se separaron eficientemente del crudo de extracción mediante cromatografia de intercambio iónico en medio ácido, debido a la carga neta introducida al derivatizar. Posteriormente fueron purificados por cromatografia en fase reversa C18 obteniendo los correspondientes derivados de epicatequina, catequina y epicatequin 3-O-galato conjugados con L-cisteína, O-etilcisteína y N-acetilcisteína. Dichos compuestos presentaron una mayor capacidad antiradicalaria que la correspondiente epicatequina mediante ensayos con los radicales libres DPPH (el cual se reduce via donación de hidrogenos y/o por transferencia electrónica) y el nuevo radical estable HNTTM sintetizado en el Departamento de Materiales Orgánicos Halogenados del IIQAB-CSIC (el cual se reduce solo por transferencia electrónica). Todos los compuestos conjugados presentaron una mayor capacidad antiradicalária que el monómero de epicatequina y los compuestos que contenian el éster de galato fueron los que generaron una mayor capacidad antiradicalária en ambos estudios. También se realizaron estudios in vitro con líneas tumorales humanas de melanoma (A375 y M21) y de colon (HT29) y con una línea no tumoral de queratinocitos humanos (HaCaT), observándose una moderada inhibición en la viabilidad celular, siendo los compuestos que contenian un éster galato los que presentaron mayor efectividad. A nivel de ciclo celular, solo los derivados que contenian el éster galato presentaron una alteración en el ciclo a concentraciones dobles de su respectiva IC50, con un aumento de la distribución en la fase S. Igualmente se observó una inducción en la apoptosis en las linias tumorales tratadas con los compuestos (siendo los conjugados con el éster galato los que presentaron una mayor inducción), pero no en la linea no tumoral de queratinocitos. Dicha inducción a la apoptosis se corroboró con otras técnicas en las que se observó condensación de cromatina y la fragmentación del ADN. Sorprendentemente, uno de los derivados que contenia un éster de galato no presentó alteración en el ciclo celular ni indujo apoptosis en las células tumorales. Igualmente, su poder antiradicalario mediante transferencia electrónica (HNTTM) fue inusitadamente bajo aun conteniendo el grupo galato. Con dichos resultados se hipotetizó la posible relación entre la transferencia electrónica y su efecto en la maquinaria celular. Asimismo, el radical estable HNTTM nos permitiria predecir de forma sencilla la capacidad pro-apoptótica de los compuestos según su capacidad de transferencia electrónica. / It has been obtained a serie of flavanol conjugates from grape byproduct through acid depolimerisation with cysteine derivatives. The conjugates were separated efficiently form the crude extract using ion exchange chromatography. The conjugates of epicatechin, catechin and epicatechin 3-O-gallate with L-cysteine, O-ethylcysteine and N-acetylcysteine were purificated by reverse phase chromatography. These conjugates showed an enhanced antiradical power using the free radical DPPH (through electron transfer and/or hydrogen atom donation) and the recently synthetized free radical HNTTM (reduced only by electron transfer). All the conjugates showed higher antiradical power than the monomeric epicatechin and the conjugates containing the gallate group were the most active compounds against free radicals. Studies about tumoral human melanoma (A375 and M21) and adenocarcinoma (HT29) cell lines and non-tumoral keratinocyte cell line (HaCaT) were also performed. It was observed a moderate effect in cell viability inhibition, being the conjugates containing the gallate group the most effective ones. Regarding cell cycle, only the conjugates containing the gallate group showed S-phase arrest to concentrations double of its IC50. Moreover, it was observed an induction of apoptosis in the tumoral cell lines treated with the conjugates, but not in the keratinocyte cell line. Surprisingly, just only one of the conjugates containing a gallate group showed a normal cell cycle distribution and it did not induce apoptosis to the tumoral cell line. Thus, its antiradical power agaisnt HNTTM (electron transfer) was lower than expected for a galloylated compound. With these results, a possible relationship between electron transfer and the cellular mechanism was proposed.

Fermentation and roasting of cocoa (Theobroma cacao) decrease levels of polyphenolic flavanol compounds. However, it is largely unknown how these changes in polyphenol levels caused by processing affect cocoa's anti-diabetic and anti-obesity bioactivities, such as inhibition of certain enzymes in the body. Polyphenol profiles, protein-binding abilities, presence of compounds termed oxidative polymers, and abilities to inhibit α-glucosidase, pancreatic α-amylase, lipase, and dipeptidyl peptidase-IV (DPP4) in vitro were compared between unfermented bean (UB), fermented bean (FB), unfermented liquor (UL), and fermented liquor (FL) cocoa extracts. Overall, there were significant decreases (p<0.05) in total polyphenols, flavanols, and anthocyanins between the two sets of unfermented and fermented cocoa extracts (CEs). All CEs effectively inhibited α-glucosidase (lowest IC50 = 90.0 ug/mL for UL) and moderately inhibited α-amylase (lowest IC50=183 ug/mL for FL), lipase (lowest IC25=65.5 ug/mL for FB), and DPP4 (lowest IC25=1585 ug/mL for FB) in dose-dependent manners. Fermentation and roasting of the samples affected inhibition of each enzyme differently (both processes enhanced α-amylase inhibition). Improved α-glucosidase and α-amylase inhibitions were correlated with presence of different classifications of oxidative polymers, suggesting that these compounds could be contributing to the bioactivities observed. Some α-glucosidase inhibition might be due to non-specific protein-binding. Improved DPP4 inhibition was strongly correlated to increased CE degree of polymerization. In conclusion, potential enzyme inhibition activities of cocoa were not necessarily negatively affected by the large polyphenol losses that occur during fermentation and roasting. Additionally, it is possible that more complex compounds could be present in cocoa that contribute to its potential anti-diabetic and anti-obesity bioactivities. / Master of Science in Life Sciences

Flavanols are polyphenols that are known to have many beneficial effects on the body. Cocoa is a major source of these flavanols. However, research on the potential anti-obesity and anti-diabetic health benefits of cocoa flavanols is lacking in the literature. Furthermore, the effect that the size of these flavanols has on the extent of its beneficial properties is also unclear. The objective of this study was to evaluate the dietary effects of cocoa flavanols on the onset of obesity, insulin resistance and impaired glucose tolerance and to determine the impact that the size of these compounds has on the magnitude of this effect. Cocoa extract was fractionated into a monomer-, an oligomer-, and a polymer-rich fraction. Six groups (n=9) of C57BL/6J mice were fed either a control low-fat diet, a control high-fat diet, or a high-fat diet supplemented with 25 mg/kg*BW of cocoa extract or one of the three cocoa fractions. After 12 weeks on these diets, the oligomer-rich fraction proved to be most effective in preventing weight gain, fat mass accumulation, elevated fasting blood glucose and impaired glucose tolerance in diet-induced obese mice. This is the first long-term feeding study to examine the relative activities of cocoa constituents on diet-induced obesity and insulin resistance. / Master of Science in Life Sciences