Source: https://www.ciencia-e-vinho.com/2018/08/
Timestamp: 2019-04-23 17:49:59+00:00

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Botrytis cinerea and Penicillium spp. (e.g. P. expanusm and P. crustosum) are the most common pathogenic agents of wine grapes (Rousseaux et al., 2014). These fungi are responsible for the production of volatile compounds that may have strong impact on the quality of wine (La Guerche et al., 2007).
Botrytis and Penicillium infect grapes are used for passito wine production during the post-harvest dehydration process (withering) (Lorenzini et al., 2016a). The incidence of their infection on withered grapes depends on culture management, seasonal conditions and withering technique. Different studies were performed to assess the B. cinerea, Penicillium spp. effects on volatile compound profile of Amarone wine, the most economically important Italian passito wine (Fedrizzi et al., 2011; Tosi et al. 2012; Zapparoli et al, 2018). Moreover, through proteomic analyses, study focused on the detection of potential markers of these fungi infection in grapes during the withering have been performed (Lorenzini, et al., 2016b; Lorenzini, et al., 2015).
In our studies, Corvina grapes were infected with B. cinerea and two Penicillum species (P. expansum and P. crustosum) in a fruit drying room of Valpolicella area. Grape samples were withered in natural conditions for about three months according traditional withering protocol. At the end of the withering process, bunches were crushed separately to obtain the grape musts for the red vinification. Must obtained from healthy Corvina grapes was used as control. Aroma compound analysis in wines obtained from healthy and infected grapes was carried out by Gas Chromatography – Mass Spectrometry (GC-MS). Sensory analysis of wines were carried out by a panel test composed by experts of Amarone wine.
Figure 1. Culture of (A) Botrytis cinerea and (B) P. crustosum and (C) P. expansum used for the grape bunches infection.
Botrytis cinerea infection of withered grapes had significant effects on Amarone wine quality. Significant changes on the content of important molecule categories such as esters, fatty acids, aldehydes and lactones were described. Similar results were reported in our previously investigations (Fedrizzi et al., 2011; Tosi et al. 2012). In particular, ethyl hexanoate, ethyl octanoate and ethyl decanoate, that play a key role in the fruity notes of wines, decreased in botrytized. Other odour-active molecules such as 1-octen-3-ol (mushroom note), phenylacetaldehyde (correlated with an oxidation-related sensory note), γ-nonalactone (coconut, sweet note) and 4-carbethoxy-γ-butyrolactone (sweet, coconut note) and N-(3-methylbutyl)-acetamide (vinegar/pungent note) increased in botrytized wine.
At sensory level, botrytized wines, tasted after one year, were perceived for more aroma complexity, almond, balsamic and resinous scent than control wine. Nevertheless, two years–aged botrytized wines were characterized by aged aroma and altered colour due to accelerated oxidation process.
Chemical and sensory analysis of wine produced from grape infected by P. expansum and P. crustosum evidenced that the fungal effects on wine quality can be important, as previously reported by Zapparoli et al. (2018). In particular, the most important odour-active molecules that varied consistently were found 3-methylthio-1-propanol (potato note), ethyl acetate (vinegar note) benzaldehydes (almond-like note), furfural (sweet, caramel scent), 4-carboxyethoxy-γ-butyrolactone, sherry lactones (oxidized note) and N-(3-methylbutyl)-acetamide. High variations in the content of these and other molecules between P. expansum and P. crustosum wines were also noted, evidencing species-related differences.
At sensory level, wine from Penicillium infected grapes were well distinguished from control wines particularly due to high intensity of pharmaceutical note giving an unpleasant aroma.
Figure 2. Infection trials. (A) Infected grape bunches of Corvina variety laid out in boxes for the withering process. Berry showing (B) B. cinerea, (C) P. crustosum and (D) P. expansum infection.
Barbara Simonato took a degree in Biological Sciences with a specialization in Food Science, at the University of Padua (Italy). She held a Ph.D. for studying the effect of technological treatment on allergenity of wheat, at the University of Padua. She is an Assistant Professor in Food Science and Technology, at the Department of Biotechnology of the University of Verona. Her research concerns the biochemistry of processed foods and their starting material.
Marilinda Lorenzini is a Ph.D. in Microbiology, at the Dipartimento di Biotecnologie of the Università degli Studi di Verona (Italy). Her research concerns the physiological, molecular and phylogenetic study of fungi (i.e. filamentous fungi and yeasts) and bacteria populations involved during the production of wines. Her research has also been concern the study of proteomic effect of filamentous fungi on grape proteome during the post-harvest withering process of grapes.
Giacomo Zapparoli is a microbiologist expert of microorganisms of grape and wine. He is an Assistant Professor in Agricultural Microbiology, at the Department of Biotechnology of the University of Verona. His research concerns the identification and taxonomy of fungi and bacteria and their effects on grape-wine quality.
Fedrizzi, F., Tosi, E., Simonato, B., Finato, F., Cipriani, M., Caramia, G., & Zapparoli, G. (2011). Food Technology and Biotechnology, 49, 529−535.
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Lorenzini, M., Millioni, R., Franchin, C., Zapparoli, G., Arrigoni, G., & Simonato, B. (2015). Food Chemistry, 179. 170–174.
Lorenzini, M., Cappello, M.S., Logrieco, A., & Zapparoli, G. (2016a). International Journal of Food Microbiology, 238, 56-62.
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Oxidative stress is caused by the insufficient capacity of biological systems to neutralize reactive species produced in excess. A serious imbalance between the generation of reactive oxygen species (ROS) and antioxidant (AOX) protection in favor of the former causes excessive oxidative damage in cells and tissues because the ROS excessive production is associated with disruption of cell cycle regulatory mechanisms (HALLIWELL, 2011).
Furthermore, excessive or prolonged ROS generation cause various health problems, such as cardiovascular disease, insulin resistance, type 2 diabetes, osteoporosis, arthritis, asthma, and inflammatory bowel disease (HALLIWELL et al., 1995; DRÖGE, 2002; RANKIN, 2004), therefore, regulation of ROS levels is critical for reducing the risk of related chronic diseases (WANG; CAO; PRIOR, 1996). Towards the end of 20th century, epidemiological studies and associated meta-analyses strongly suggested that long term consumption of diets rich in plant polyphenols offered some protection against development of cancers, and neurodegenerative diseases.
The phenolic compounds in grapes and derivatives, mainly the flavonoids, flavanols, flavonols and anthocyanins, are associated with improved health, along with other compounds which are not flavonoids, such as phenolic acids and the stilbene resveratrol (SAUTTER, et al., 2005; KRIKORIAN et al., 2012). Besides these functions, the chemical structure of polyphenols, mainly flavonoids and stilbenes (resveratrol), makes them suitable to act as antioxidants, trapping and neutralizing free radicals.
Cell culture has often been used to study the cellular effects of reactive species and of antioxidants, and many useful data have resulted (HALLIWELL, 2011). Hydrogen peroxide is a physiological constituent of living cells and is continuously produced via diverse cellular pathways. Intracellular steady-state concentrations of H2O2 above 1 μM are considered to cause oxidative stress inducing growth arrest and cell death (STONE; YANG, 2006). In experimental models used to investigate physiological functions and toxic effects of H2O2, oxidative stress responses of cells, or cryoprotection by antioxidant agents, cultured cells are often exposed to H2O2 added as a bolus into the culture medium (GÜLDEN et al., 2010).
To determine wine could protect against oxidative stress-induced cell death, the SH-SY5Y cell line was used as an in vitro model and H2O2 as pro-oxidant insult. After 24 h of H2O2 exposure in combination with wine at 250 μg/mL significantly increase the cell viability in the absence of H2O2, while 250 μg/mL of wine protected against cytotoxicity. Similar results were found by Xiang et al. (2014), when SH-SY5Y cells were treated with 100 µM H2O2 with 4 mg/mL red wine extracts or red wine adding 10-fold resveratrol, all wine varieties showed significant neuroprotective effect against H2O2-induced oxidative stress. Results of the intracellular antioxidant capacity measurements of the wine samples using the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay in wine had a pro-oxidant effect per se by increasing the dichlorofluorescein (DCF) levels in the absence of H2O2, whereas, 250 and 500 µg/mL of wine significantly reduced H2O2-induced production of ROS. Results obtained suggest that wine is a potential antioxidant and have positive effect against reactive species generated in SH-SY5Y cells, suggesting a neuroprotective effect.
Cristiane Copetti. Graduated in Nutrition by Franciscana University (Brazil) in 2007. Master in Food Science – Food Chemistry and Biochemistry by Federal University of Santa Catarina (Brazil) in 2010. Professor of Nutrition in Franciscana University between 2010 and 2012. Ph.D. in Food Science and Technology - Processing and Analysis of Wine and Other Alcoholic Beverages by Federal University of Santa Maria (Brazil) in 2017, during this period, the research activity was divided in laboratories of Center of Oxidative Stress Research (CEEO), Department of Biochemistry and Institute of Food Science and Technology (ICTA) of Federal University of Rio Grande do Sul. Currently works in the Criciúma city (Brazil) composing the team federal program of the Family Health and Basic Care Support Center and visiting professor of cultural gastronomy course of Federal University of Rio Grande do Sul (Brazil).
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Grape accumulates numerous polyphenols with abundant health benefit and organoleptic properties that in planta act as key components of the plant defense system against diseases. Besides ubiquitous phenolic compounds including flavonoids, hydroxycinnamates and benzoates, grapevine accumulated peculiar non-flavonoid compounds called stilbenoids. Grapevine accumulates stilbenoids mainly in lignified organs. By the way, the winter-pruned wood from vineyard has been proposed as a source of valuable bioactive compounds (Houillé et al 2014; 2015a; 2015b; Billet et al., 2018a).
Metabolomics is the systematic study of all small-molecular weight metabolites present within a cell, tissue or organism. Grape metabolomics has contributed greatly to the chemical characterization of wine (Flamini et al., 2013). Recently, the development of metabolomic approaches has led to the concept of metabolic phenotype that considers a metabolic profile as a result of the genotype expression under environmental conditions. It is particularly relevant for plant metabotypes based on specialized metabolites since they might vary according biotic and abiotic stresses.
In the present study (Billet et al., 2018b), a field experiment (Figure 1) was setting up with uniform pedo-climatic factors and viticultural practices of growing vines to favor the genetic determinism of polyphenol expression. We used UPLC-MS-based targeted metabolomics to identify the metabolic composition of grape stem that correspond to the main site of accumulation for stilbenoids in vines. Chemometric tools including principal component analysis (PCA), partial least square discriminant analysis (PLS-DA) and hierarchical cluster analysis (HCA) were used to identify the overall metabolomic similarity/dissimilarity among the samples. Metabolomic and SSR-based clusterings were compared to assess the relationship between biochemical and genetic distances.
Figure 1. Location of the vineyard plots planted with the 8 different grape varieties in Amboise (Loire Valley, France). Grape stems were randomly pruned across the total area of each plot as indicated by circle positions.
As a result, metabolic profiles of grape stems enabled discrimination of varietal origin suggesting that polyphenol profiles exhibit variety-dependent metabolic fingerprints (Figure 2). HCA perfectly separated genotypic replicates but also grouped genotypes according to their biochemical distances, thus suggesting a classification in accordance with their genetic distance.
A first group of metabotypes (Gamay, Chardonnay, Pinot Noir) corresponds to closely related genotypes previously grouped called “Noirien” when defined as eco-geographical groups by ampelographers. A second group of metabotypes (Chenin, Sauvignon) belongs to the group called “Messile” originated in the French Loire Valley. Recently, these parentage relationships were confirmed by DNA sequence studies using SSR and SNP markers. Chardonnay and Gamay are full-siblings of Pinot noir and Gouais Blanc and Chenin is a possible natural cross of between Sauvignon and Savagnin.
Field-based metabolomic experiments may reflect real growth conditions, however such investigations are challenging because the metabotype reflects both genetic and environmental conditions. In the present field experiment, the genotypic discrimination was achieved using a harvest protocol that limits the effect of environmental conditions and highlights variations due to the genotype. Because, polyphenol content in grape stems might be locally induced in response to pathogen attacks, the quality of sampling design in the vineyard is determinant to limit intra-plot variations.
Dealing with the different sources of variability in real growing conditions (genotypes, environment and management interventions) constitutes one of the future challenges for field-omics. In a near future, the present polyphenol metabotyping approach coupled to multivariate statistical analyses might assist grape selection programs to improve metabolites with health-benefit potential and plant defense traits.
Figure 2. metabolic profiles of grape stems.
Dr. Arnaud Lanoue is a phytochemical analyst. He obtained a joint Ph.D. at the Universities of Amiens (France) and Geneva (Switzerland) in 2002 on the bioproduction of plant specialized metabolites. He conducted postdoctoral research at the Juelich Research Center (Germany) on plant natural products as signaling molecules within the Biorhiz project (Marie Curie actions-Research Training Networks). Since 2007, he is Associate Professor at the Faculty of Pharmacy in the University of Tours (France) in the Lab “Biomolecules and Plant Biotechnologies” (@BBVEA2106_Tours, Plant & Biotech Lab - BBV Ea2106 Tours) where is leading several research programs on the valorization of grape polyphenols.
Billet, K., Houille, B., Dugé de Bernonville, T., Besseau, S., Oudin, A., Courdavault, V., et al. (2018b). Field-based metabolomics of Vitis vinifera L. Stems Provides New Insights for Genotype Discrimination and Polyphenol Metabolism Structuring. Front. Plant Sci. doi: 10.3389/fpls.2018.00798.
Flamini, R., De Rosso, M., De Marchi, F., Dalla Vedova, A., Panighel, A., Gardiman, M., et al. (2013). An innovative approach to grape metabolomics: stilbene profiling by suspect screening analysis. Metabolomics 9(6), 1243-1253. doi: 10.1007/s11306-013-0530-0.
The anthocyanins are responsible for most of the red, blue and purple colors of fruits and flowers, in addition to some vegetables such as purple varieties of potatoes, corn, beans, onions and carrots, or red cabbage and some plant leaves such as purple trees, new foliage or the red leaves around poinsettia flowers or even autumn leaves (Figure 1). Although thousands of different anthocyanins have been characterized, almost all can be classified into six basic structural types that differ only in the number of hydroxy or methoxy substituents in the B ring (Scheme 1). In nature, the 3-hydroxy group of anthocyanins is always glycosylated, which is apparently important for thermal stability. Anthocyanins derived from malvidin-3-O-glucoside (Scheme 2) typically predominate in Vitis vinifera grapes, with lesser amounts of anthocyanins derived from cyanindin, peonidin, delphinidin and petunidin.
Scheme 1. The six most common basic chemical structures of anthocyanins, shown as the fully deglycosylated or aglycone forms, referred to as anthocyanidins; the percentages in parentheses represent the approximate relative natural abundances in plants.
Anthocyanins accumulate in vacuoles of plants and have been proposed to play at least three important biological roles in plants: (1) as a color signal to attract pollinators to flowers or frugivores to fruit; (2) as an antioxidant; and (3) in the case of leaves, photoprotection of the photosynthetic apparatus against excess solar radiation. Although anthocyanins are safe to use as food colorants, with desirable antioxidant properties and other potential health benefits, their color is restricted to relatively acidic solutions (pH < 3). Especially around neutral pH, anthocyanins lose most of their color due to a pH-dependent reaction with water and a series of subsequent structural transformations.3 In this regard, red wines have managed to improve on Nature by slowly transforming the anthocyanins initially present in grape juice into a variety of more complex pigment molecules during the ageing of the wine. These complex reactions of the anthocyanins with colorless molecules and yeast metabolites present in the wine produce pyranoanthocyanins (Scheme 2), which are important contributors to the final color of the wine. Pyranoanthocyanins differ from anthocyanins by the presence of an additional pyran ring between the 4-carbon and the 5-hydroxy group of the anthocyanin precursor, which blocks the reaction with water, making the color of mature wines more pH-stable and much less susceptible to bleaching by additives such as sulfite than that of young wines. Pyranoanthocyanins also appear to contribute to the radical scavenging antioxidant capacity and to the organoleptic properties of aged red wines.
Scheme 2. Generic reaction for the formation of a Vitisin B type pyranoanthocyanin via a reaction of malvidin 3-O-glucoside (oenin), the predominant anthocyanin in Vitis vinifera grapes, with the enol form of a copigment during the maturation of red wine.
Scheme 4. Excited state proton transfer process of malvidin 3-O-glucoside. Absorption of light (h) by the ground state cation form AH+ produces the first excited singlet state of the cation, [AH+]*1, which transfers a proton to water to form the excited singlet state of the conjugate base, A*, in about 20 ps. The excited base form A* lives on average ca. 200 ps before transforming the excitation energy into heat and returning to the ground state of the base, A, which then reprotonates back to AH+ with no net chemistry.
Anthocyanins also form complexes with colorless electron-rich “copigments” molecules such as hydroxybenzoic or hydroxycinnamic acids. Intramolecular copigmentation complexes are also present in acyl anthocyanins with one or more copigment molecules covalently attached to the sugar residues. The charge-transfer interactions involved in the copigmentation not only help stabilize the cationic form of the anthocyanin chemically, but also open up a new charge-transfer mediated channel for direct conversion of the absorbed light energy into heat that is even faster ( 1 ps) than excited state proton transfer. As far as we know, there are no published studies of copigmentation of pyranoanthocyanins, but it is reasonable to expect that it should occur.
Dr. Frank Herbert Quina (quina@usp.br) is a full professor at the Instituto de Química, Universidade de São Paulo, in São Paulo, Brazil. He received his Ph.D. from CALTECH (1973) and immigrated to Brazil in 1975 after a post-doctoral stint at the University of North Carolina, Chapel Hill. He is a Level 1A research fellow of the Brazilian National Research Council (CNPq), a fellow of the Royal Society of Chemistry, IUPAC and the Inter-American Photochemical Society, and a member of the Brazilian Academy of Sciences. He was a Senior Editor of Langmuir (2013-2016) and is currently an Associate Editor of ACS Omega and on the editorial boards of Photochemical and Photobiological Sciences, Photochemistry and Photobiology and the Brazilian Journal of Chemical Engineering. His group maintains active international research collaborations with groups in Portugal, Chile, Canada, China and the USA in the areas of the chemistry and photochemistry of natural plant pigments and the structure and dynamics of self-assembling colloidal systems. He is the fifth generation of his family with a registered cattle brand and the third generation to grow tropical fruit.

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