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Oak barrel tannin and toasting temperature: Effects on red wine anthocyanin chemistry
Accepted Manuscript Oak barrel tannin and toasting temperature: Effects on red wine anthocyanin chemistry Aude A. Watrelot, Andrew L. Waterhouse PII:
S0023-6438(18)30757-6
DOI:
10.1016/j.lwt.2018.09.025
Reference:
YFSTL 7405
To appear in:
LWT - Food Science and Technology
Received Date: 31 May 2018 Revised Date:
29 August 2018
Accepted Date: 11 September 2018
Please cite this article as: Watrelot, A.A., Waterhouse, A.L., Oak barrel tannin and toasting temperature: Effects on red wine anthocyanin chemistry, LWT - Food Science and Technology (2018), doi: https:// doi.org/10.1016/j.lwt.2018.09.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Oak barrel tannin and toasting temperature: Effects on red wine anthocyanin chemistry.
3 Aude A. Watrelot*, Andrew L. Waterhouse
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Department of Viticulture and Enology, University of California Davis, One Shields Ave., Davis, CA 95616-5270, USA
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*corresponding author:
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A.A. Watrelot
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Department of Viticulture and Enology, University of California Davis, One Shields
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Ave., Davis, CA 95616-5270, USA
Telephone: +1-530-752-5054
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Fax: +1-530-752-0382 E-mail: [email protected]; [email protected]
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Abstract Oak-derived compounds are generally considered to be important in red wine
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perception and color qualities. In this study, the percentage of loss and kinetic rates of
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degradation of monomeric anthocyanins as well as the formation of pigmented tannins were
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monitored in red wines aged for 8 and 12 months in oak barrels differentiated by stave
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ellagitannin content (low LTP, medium MTP and high HTP) and toasting level (160 and 180
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°C as initial temperature). The malvidin coumaroyl glucoside is found to be the most reactive
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monomeric anthocyanin during barrel aging. While color intensity was not affected, the
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results indicated that in presence of high ellagitannin content red wines exhibit a lower loss
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and slower degradation of monomeric anthocyanins and slower formation of pigmented
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tannins.
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Keywords Monomeric
anthocyanins,
kinetic
rates,
wood
toasting
level,
ellagitannin
concentration, pigmented tannins
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1. Introduction
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Anthocyanins are the most important pigments (of the Greek anthos = flower and kianos =
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blue) of the vascular plants, present in vacuoles. These pigments are water-soluble and
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responsible for pink to blue color range in flowers and fruits. The structure of anthocyanidins
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ACCEPTED MANUSCRIPT (anthocyanin aglycones) is a 2-phenyl-1-benzopyrylium nucleus diversely substituted by OH
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and OMe groups, especially on the B-ring. The pigment color depends on several factors,
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such as substitution on the B-ring, pH, presence of copigments and metal ions (Dangles,
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Stoeckel, Wigand, & Brouillard, 1992). Anthocyanins found in red wine (mostly malvidin-3-
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O-glucoside) are extracted from grapes during winemaking process and are able to interact
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with cell wall material (Buchweitz, Nagel, Carle, & Kammerer, 2012) but also with
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acetaldehyde, pyruvic acid, condensed tannins and hydrolysable tannins to form new products
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such as vitisin A and B, anthocyanin-flavanol, and anthocyanin-ellagitannin allowing color
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stabilization (Chassaing et al., 2010; Mateus & de Freitas, 2001).
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Ellagitannins are hydrolysable tannins from wood that are easily extracted by wine that is
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a hydroalcoholic solution. Due to their chemical structure they are known to be the first
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barrier against phenolic compounds oxidation but ellagitannins can be degraded during the
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barrel processing, especially toasting. Higher toasting temperatures and the duration of the
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toasting process lead to higher degradation of ellagitannins, (Chira & Teissedre, 2015;
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Fernández de Simón, Cadahía, del Álamo, & Nevares, 2010). Many studies have focused on
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the color properties of red wine aged in oak barrels or in stainless tanks with oak chips and on
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the formation of new pigments involving anthocyanins that could explain the color
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stabilization (De Rosso, Panighel, Dalla Vedova, Stella, & Flamini, 2009; Dumitriu, Lerma,
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Cotea, Zamfir, & Peinado, 2016; Ortega-Heras, Pérez-Magariño, Cano-Mozo, & González-
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San José, 2010). The addition of wood chips during wine aging induced a higher stabilization
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of color and various level of degradation of monomeric anthocyanins depending on the type
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of wood, due to their porosity that lead to different rate of ellagitannins extraction (Gambuti,
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Capuano, Lisanti, Strollo, & Moio, 2010; Kyraleou et al., 2016). However few investigations
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have addressed the effect of oak barrel ellagitannin levels or toasting temperature on the fate
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of anthocyanins (Mateus & de Freitas, 2001). Moreover, the use of enological tannins in red
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tannins are not well characterized and their effects are controversial. In many articles,
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enological tannins contribute to red wine color stabilization and mouthfeel perception
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(Bautista-Ortin, Cano-Lechuga, Ruiz-Garcia, & Gomez-Plaza, 2014; Obreque-Slier, Peña-
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Neira, & López-Solís, 2012), however other studies show that the addition of enological
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tannins to red wine does not have a significant effect and/or has a negative effect on red wine
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quality (Harbertson, Parpinello, Heymann, & Downey, 2012). In our previous article
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(Watrelot et al., 2018), the effect of oak barrel tannin and toasting temperature on condensed
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tannin chemistry has been elucidated and some data such as ellagic acid content and the
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percentage of pigmented tannins has been discussed . The aim of this work is to complement
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the previous one, investigating the effect of the level of oak barrel tannin and toasting
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temperature on the degradation rates of wine anthocyanins and on the formation of pigmented
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tannins.
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2. Material and Methods
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2.1. Reagent and chemicals
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All solvents were HPLC grade. Acetonitrile, methanol, acetic acid, L- (+) ascorbic
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acid, o-phosphoric acid and ammonium dihydrogen phosphate were purchased from VWR
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International (Radnor, PA). (-)-epicatechin, (+)-catechin hydrate and ellagic acid were
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purchased from Sigma-Aldrich (St. Louis, MO). Oenin chloride was purchased from
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Extrasynthèse (Lyon, France).
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2.2. Oak barrel characteristics
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The barrels (225 L) were made from oak (Q. sessilis) coming from French forests (90
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coopering.
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2.2.1. Oak wood sorting methodology according to its tannin potential (TP)
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Wood classification according to its ellagitannin content was performed by near-infrared
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(NIR) spectroscopy using a technique based on the use of an acousto-optic tunable filter
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(AOTF, Brimrose, USA) (Watrelot et al., 2018). Briefly, after machining, the untoasted staves
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were first being intended to gather spectral data by NIR and then ellagitannins total level
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analyzed through ellagic acid dosage in HPLC-DAD after extraction and hydrolysis in acidic
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medium. The calibration was performed from a partial least squares (PLS) regression after
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selecting the most discriminant spectral zones. The correlation coefficient between spectral
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measurement and HPLC dosage of total ellagitannins (0.89) shows the performance of the
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model used. The calibration has been carried out on 450 samples from 27 different groups.
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The classification from the NIR method was used to sort the staves prior to toasting into three
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groups of tannin potential (TP) i.e. the ellagitannin content in untoasted wood: Low or LTP <
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4000, Medium or MTP from 4001 to 6000 and High or HTP from 6001 to 8000 µg of ellagic
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acid equivalent / g of dry wood.
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2.2.2. Wood toasting methodology
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Toasting was conducted after automated steam bending (4 min), which yields a neutral
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untoasted barrel. Toasting, controlled by computer, was performed using radiant heat rather
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than direct contact with flame. The toasting pot was fed regularly with fuel (100 % oak
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pellets) by an auger. The barrel, placed on a turntable, rotates around a double cone that
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covers the fire and channels the heat source, during the entire toasting phase. An infrared
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sensor, performing measurements on the internal surface of the shell, provides temperature
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control, with a heating accuracy of +/- 3 ° C. In our study, all barrels (LTP, MTP and HTP)
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initial temperatures were compared (160 and 180 °C), as previously described by Watrelot et
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al., 2018.
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2.3. Red wine winemaking
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Red wines were made by two Californian wineries (B and CR) and one winery from
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Washington State (CW) from 2015 Cabernet Sauvignon grapes of Napa Valley and Wahluke
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slope, respectively. The winemaking process of B and CR is already described in Watrelot et
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al., 2018. Briefly, the alcoholic fermentation was made using Zymaflore F15 and D254 yeast
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strains in B and CR wines, respectively. Spontaneous malolactic fermentations occurred after
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yeast fermentation was complete. Sulfur dioxide, 60 mg/L, was added to the wines prior to
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barreling and three wine samples of each winery were analyzed. The CW wine was made
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from grapes harvested by the beginning of October 2015. The wine was fermented for 5 days
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at 26.7 °C with Red Start Premier Cuvee yeast strain. Following inoculation with Oenococcus
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oeni, the malolactic fermentation of CW wine in tank took 10 days prior to barreling. 60 mg/L
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of oak flour (Innerstave Oak plus flour) was added into the blend prior to barreling. Each wine
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was barreled in 18 barrels (225 L) (three barrels for each 6 modalities previously described).
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Barrels were topped every month, racked every three months and wine samples were analyzed
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after each racking time. B and CW wines were aged for 12 months in 18 barrels (triplicate of
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the 6 barrel modalities previously described) and CR wines were aged for 8 months in 18
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barrels (triplicate of the 6 barrel modalities previously described) as decided by the winery
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involved in this study (Figure 1).
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2.4. Red wine characterization
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2.4.1. Sampling
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For each winery, three wine samples were collected before barreling (named T0) and 6
ACCEPTED MANUSCRIPT chemically analyzed. The wine was then barreled in triplicate in 6 oak barrel modalities (LTP-
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160, LTP-180, MTP-160, MTP-180, HTP-160 and HTP-180) and aged for 3, 5, 8 and 12
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months for wine from B and CW winery. After 3 months of aging, three wine samples were
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collected from each oak barrel modalities (named T3), corresponding to 18 samples for each
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winery. The same sampling was carried out after 5, 8 and 12 months of aging (T5, T8 and
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T12). CR wines were aged for 8 months and the sampling was carried out on wines after 3, 5
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and 8 months of aging. To summarize, a total of 18 wine samples per winery at each aging
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time was analyzed (Figure 1). pH, ethanol content and titratable acidity were determined
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using a Winescan FT120 (FOSS, Eden Prairie, Minnesota, USA) for each modality and
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replicate and the average of the eighteen wines was used to compare these parameters
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between wineries. At the end of aging, the pH was 3.6, 3.7 and 3.8 in CR, B and CW wines,
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respectively; the ethanol content was 14.9, 15.2 and 14.7 % vol in CR, B and CW wines,
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respectively and the titratable acidity was 6.0, 6.0 and 5.3 g/L in CR, B and CW wines,
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respectively.
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2.4.2. Ellagic acid concentration
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The total ellagitannin concentration of red wine was determined by the quantification
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of ellagic acid released after acidic hydrolysis and HPLC quantification, as previously
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described (Watrelot et al., 2018). In this study, the ellagic acid concentration of CW wines at
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each sampling time and of B and CR wines only at the end of aging are shown. The ellagic
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acid concentration in B and CR wines at each sampling time is available in a previously
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published article (Watrelot et al., 2018).
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2.4.3. Monomeric anthocyanins
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Monomeric anthocyanins of red wines were characterized by C18 reversed-phase
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HPLC-DAD after direct injection of 20µL filtered wine (0.45µm, PTFE filters) according to 7
ACCEPTED MANUSCRIPT (Ritchey & Waterhouse, 1999). The 1100 Agilent HPLC system was composed of a degasser,
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an autosampler, a quaternary pump and a diode array detector, controlled by Chemstation
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software. For analysis, a reverse phase 250 × 4 mm, 5µm, RP-18 Lichrospher® 100 column
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was used. The mobile phases used were composed of solvent A (50 mM ammonium
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dihydrogen phosphate pH 2.6), solvent B (20% solvent A in acetonitrile) and solvent C (0.2
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M ortho-phosphoric acid in water, pH1.5) with the following elution gradient: 0 min (0 %B; 0
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%C), 8 min (8 %B; 0 %C), 20 min (14 %B; 86 %C), 25 min (16.5 %B; 82 %C), 35 min (21.5
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%B; 78.5 %C), 70 min (50 %B; 50 %C), 75 min (0 %B; 0 %C), 90 min (0 %B; 0 %C). The
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flow rate was set at 0.5 mL/min and the column temperature at 40 °C. Detection was made at
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520 nm. Monomeric anthocyanin concentration is expressed in malvidin-3-O-glucoside
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equivalents.
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2.4.4. Pigmented tannins, color intensity and hue
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The percentage of pigmented tannins was measured by gel permeation chromatography and
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corresponded to the ratio of absorbance at 520 nm to 280 nm, as previously described
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(Watrelot et al., 2018). In this study, only the percentage of pigmented tannins in CW wines is
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presented. These values in B and CR wines can be found in Watrelot et al. (2018). The
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spectrum of all red wines at the end of aging was determined using 10 mm polymethyl
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methacrylate (PMMA) cuvettes by a UV-Visible spectrophotometer. The absorbance values at
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420 nm, 520 nm and 620 nm were analyzed to determine the color intensity (sum of the
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absorbances at these three wavelengths) and the hue (420 nm: 520 nm ratio) of red wines for
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the three wineries and all oak barrel modalities. The analyses were done for each wine
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(experimental triplicates).
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2.5. Statistical analysis Experiments were performed in triplicate: red wine was aged in three barrels for each 8
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expressed as the mean and the standard deviation of experimental replicates and all statistical
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analysis were made using XLstat software for Microsoft Excel®. One way-ANOVA using a
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Fisher (LSD) test with a confidence interval of 95 % was carried out on all samples to identify
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significant differences between all barrel modalities for each winery. A multivariate analysis
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of variance (MANOVA) using toasting temperature and ellagitannin content as factors was
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performed to determine the effect of these individual factors and their interactions on the
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monomeric anthocyanin loss percentage and their degradation rates for all three wineries.
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3. Results and Discussion
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3.1. Ellagic acid concentration
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Ellagitannins from wood are extractable compounds in wine due to the hydroalcoholic
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medium. As previously observed in B and CR wines (Watrelot et al., 2018), the extraction of
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ellagitannin into CW red wine increased with aging (Figure 2), even though this wine had
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some ellagitannins added prior to barreling (T0). The increase of ellagic acid concentrations
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in the red wines between oak barrel modalities were the same up to 8 months of aging, as
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previously observed, and were not significantly different at the end of aging (12 months). This
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is due to the slow extraction for a few months after wine contact and to an equilibrium that
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probably occur between ellagitannins already and still extracted (Michel et al., 2011). After 8
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months of aging, ellagic acid concentration was the highest in red wine aged in oak barrel rich
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in ellagitannin and toasted at low temperature (HTP-160) (Figure 2) and was the lowest in red
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wine aged in oak barrel with low ellagitannin content and toasted at higher temperature (LTP-
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180). In accordance with Michel, Jourdes, Giordanengo, Mourey, & Teissedre, (2012), staves
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rich in ellagitannin (HTP) release higher ellagitannin content in wine. Higher ellagitannin
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ellagitannins and other wood compounds are degraded by high temperature (Chira &
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Teissedre, 2015).
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3.2. Monomeric anthocyanins degradation of red wine aged in oak barrels
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Large variation of monomeric anthocyanin concentrations was observed between the
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three wines aged in oak barrels (Table 1). As expected, malvidin-3-O-glucoside was the most
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abundant followed by the acetylated form of malvidin-3-O-glucoside. Prior to barreling (T0),
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CR wines were richer in total monomeric anthocyanin (425.1 mg/L as malvidin-3-O-
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glucoside eq.) than B and CW wines (245.8 and 190.8 mg/L as malvidin-3-O-glucoside eq.,
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respectively) which can be related to the initial anthocyanin concentration in grapes and to the
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winemaking process. The concentrations of each monomeric anthocyanin are in the typical
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range of concentrations found in red wines from Cabernet sauvignon grapes (Monagas,
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Gómez-Cordovés, & Bartolomé, 2005). At the end of barrel aging, the concentration of each
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monomeric anthocyanin in B and CR wines was similar, except delphinidin-3-glucoside and
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petunidin-3-glucoside were slightly higher in CR wines.
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The percentages of loss of monomeric anthocyanins during red wine aging in the oak
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barrels toasted at two temperatures and of three levels of ellagitannin are shown in Table 2. In
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accordance with the concentrations of anthocyanins prior to barreling and at the end of aging
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(Table 1), the percentage of loss of total monomeric anthocyanin was higher in CR wines than
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CW and B wines. This indicated that the anthocyanin content of the CR wines was less stable
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during aging. This wine had the highest concentration of condensed tannins in the wine at the
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end of aging (943 mg/L as catechin equivalent (Watrelot et al., 2018)), and we expect these
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tannins react with anthocyanin, leading to the loss of monomeric anthocyanins, as observed
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by others (García-Estévez et al., 2013). In spite of having the highest concentration in red
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degraded during aging. Whatever the aging modalities, the percentage of loss of monomeric
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anthocyanin followed this range: Malvidin-3-O-(6''p-coumaroyl)-glucoside > peonidin-3-
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glucoside > malvidin-3-O-(6''-acetyl)-glucoside > malvidin-3-O-glucoside > delphinidin-3-
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glucoside ≥ petunidin-3-glucoside, following a pattern observed in Port wines (Mateus & de
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Freitas, 2001) as well as in Cabernet Sauvignon and Tempranillo wines (Monagas et al.,
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2005). The percentage of loss of malvidin-3-O-glucoside, delphinidin-3-glucoside and
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petunidin-3-glucoside was very similar (significantly identified but not shown) suggesting
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that the number of hydroxylation or methoxylation of the three groups on the B-ring does not
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play a major role in their degradation, as observed in blueberry juice (Howard, Brownmiller,
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Mauromoustakos, & Prior, 2016). However, the percentage of loss of peonidin-3-glucoside
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that is an anthocyanin with 1 hydroxyl group and 1 methoxyl group on the B-ring was much
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higher than the anthocyanin with 3 groups on the B-ring, suggesting that the number of
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groups on the B-ring is involved in the stability of the monomeric anthocyanins (Castañeda-
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Ovando, Pacheco-Hernández, Páez-Hernández, Rodríguez, & Galán-Vidal, 2009). As the
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level of glycosylation of the anthocyanin, the type of acetylation showed an effect on the loss
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of anthocyanin, for which the coumaroyl malvidin was more degraded than the acetylated
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malvidin (Mateus & de Freitas, 2001; Monagas et al., 2005). It is possible that the loss of the
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acylated anthocyanins was due to ester hydrolysis of the acyl group or to precipitation of it
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(Gil et al., 2017) and not oxidation of the anthocyanidin. The effect of the oak barrel
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modalities on the loss of monomeric anthocyanins varied depending on the red wine. B and
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CW wines did not show any significant effect of the toasting temperature (Table 2) in contrast
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to CR wines where 180 °C significantly increased the percentage of loss of all monomeric
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anthocyanins except delphidin-3-glucoside, compared to 160 °C. This effect was significant
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only in CR wines probably due to the higher total monomeric anthocyanin concentration
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concentration in red wines, that can limit their barrier effect against oxidation, leading
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therefore to the loss of anthocyanins. This agrees with Gonzalez-Saiz et al. (2014) who
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showed that the content of anthocyanins decreased in wine that infused with medium to
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medium-plus toasted wood chips.
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For all wines, the level of ellagitannin in staves showed significant effect on the loss of
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anthocyanins (Figure 2 and Table 2). Wine aged in low ellagitannin content (LTP) barrels
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showed a higher percentage of loss than in medium and high ellagitannin content, and
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therefore contained significantly less monomeric anthocyanins at 12 months. This showed
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once again that ellagitannins in wine from barrel are able to protect the anthocyanins from
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their degradation by oxidation during aging, and not necessary interact with them to form new
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stable pigment (Chassaing et al., 2010). The combined effect of toasting and ellagitannin
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content in staves was significant only in CR wines for malvidin-3-O-glucoside, malvidin-3-O-
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(6''-acetyl)-glucoside and the petunidin-3-glucoside (Table 2). Wine aged in LTP-180 barrels
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that contained the least ellagitannins induced the highest loss of anthocyanins in contrast to
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HTP-160 that contained the highest ellagitannin content.
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In agreement with some studies (Bakker, 2015; Mateus & de Freitas, 2001; Oliveira,
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Barros, Silva Ferreira, & Silva, 2015), the degradation reaction of anthocyanins follows first-
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order kinetics. The rate constants (k) were calculated from the slope after linear regression of
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ln (C/C0) = f(t), where C is the concentration of anthocyanin, C0 is the concentration of
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anthocyanin prior barreling and t is the aging time in months (Figure 1 in supplementary
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information). The rate constant varied depending on the anthocyanin structure as well as on
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the oak barrel modality (Table 3). Like the percentage of loss, the rate constant of degradation
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was higher for malvidin-3-O-(6''p-coumaroyl)-glucoside and was the lowest for delphinidin-
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3-glucoside, for all three red wines (B, CR and CW). The degradation rate constant of
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malvidin-3-O-glucoside was lower than those of acylated malvidin derivatives (malvidin-3-O-
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6’’-acetyl-glucoside and malvidin-3-O-6’’-coumaroyl-glucoside), in accord with Monagas et
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al., (2005) and Mateus & de Freitas, (2001). As noted above, the loss of acylated
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anthocyanins likely involves ester hydrolysis as well as oxidation.
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components are fairly small, the products, the simple glycosides, would not increase by
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noticeable amount. Between the three wines, the rate constant of total anthocyanin loss varied
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from 0.041 to 0.101 months-1 in B and CR, respectively. B and CW wines showed similar rate
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constants even if B had the lowest values, while in CR wines the rate constants were much
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higher. The kinetic rate constants were significantly higher in CR and CW wines aged in oak
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barrel toasted at 180 °C than at 160 °C (Table 3), revealing that the toasting temperature of
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oak barrel affects the kinetics of degradation of monomeric anthocyanins. As explained
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above, the higher thermolytic degradation of ellagitannins from wood during the toasting
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process at 180 °C (initial toasting temperature) (Chira & Teissedre, 2015) reduces their
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protective effect against anthocyanin degradation.
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In the three wines, the rate constants for all anthocyanins were significantly affected
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by the ellagitannin content in staves, where higher k values were observed in wines aged in
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LTP barrels. This confirms the above observation where low ellagitannin content (due to
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thermolytic degradation or due to low initial concentration in wood) induced a faster loss of
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monomeric anthocyanins due to the lack of compounds protecting against oxidation reactions.
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Wine aged in LTP-180 barrels showed a faster loss (higher k value) compared to wine aged in
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HTP-160 (which contained the highest concentration of ellagitannins). The loss of monomeric
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anthocyanins is usually associated with the formation of anthocyanin-pyruvic acid adducts
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(vitisin A) (Monagas et al., 2005), but this compound was not found in our samples probably
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because the sensitivity of detection of our method was too low.
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As reported earlier, the percentage of pigmented tannins at the end of aging was 13
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respectively), which might be related to the highest loss percentage and the fastest
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degradation of monomeric anthocyanin in wines from CR winery. The highest concentration
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of pigmented tannins (Figure 3 and Watrelot et al., 2018) was observed in wine aged in LTP-
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180 and the lowest in HTP-160, suggesting that in presence of a low concentration of
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ellagitannins, the monomeric anthocyanins are more able to interact with condensed tannins to
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form pigmented tannins. As suggested by Gambuti et al., (2017), the oxidation occurring
315
during red wine aging responsible for tannin chemical reactions is a key parameter in the
316
formation of pigmented tannins, but also the phenolic structure. The tannins: anthocyanins (T:
317
A) ratio might be an indicator of the formation of pigmented tannins that is supposed to
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correlate with the formation of pigmented tannins identified above. In our study, the T: A
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ratio, based on tannin and anthocyanin concentrations at the end of aging, was higher in LTP-
320
180 wines from CR and CW wines, while for all wines, the T: A ratio was lower in HTP-160
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wines (data not shown), which was in agreement with the formation of pigmented tannins
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observed. As previously reported by Boulton, (2001), conversion of monomeric anthocyanins
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to pigmented tannins during aging might be responsible for stable red wine color. In order to
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assess this hypothesis, the color intensity and hue of all red wines at the end of aging were
325
measured, based on their UV-Vis spectra (Figure 4). No clear significant effect was observed
326
in term of color intensity (Figure 4A) and hue (Figure 4B) for all wines, regarding oak barrel
327
modalities. In B wines, the color intensity seemed to be higher in LTP-180 wines, but this was
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not observed in other wineries. As previously explained by Harbertson, et al., (2012) the
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addition of enological tannins to wine does not always provide an improvement of the
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mouthfeel or color. In this study, the lack of influence of oak barrel ellagitannin levels on red
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wine color might be attributed to the loss of anthocyanins combined with the formation of
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new pigments.
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ACCEPTED MANUSCRIPT While little effect of oak barrel ellagitannin content on red wine color intensity was
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observed, oak barrels rich in ellagitannins led to a slower and lesser loss of monomeric
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anthocyanins. These barrels were also associated with a lesser formation of pigmented
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tannins.
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The authors thank the three wineries from California and Washington State involved in
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4. Acknowledgments
this project for providing red wines.
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This study was funded by Vicard Generation 7, France.
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Table captions. Table 1. Concentrations of monomeric anthocyanins (expressed in mg/L malvidin-3-Oglucoside) in red wine prior to barreling (T0) and after aging in oak barrels (B, CR, CW wines).
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Table 2. Percentage of loss of monomeric anthocyanins in red wine aged for 12 (B and CW) and 8 months (CR) in oak barrel toasted at two initial temperatures (160 and 180 °C) and with staves of three levels of ellagitannin (LTP, MTP and HTP). Multivariate analysis of variance of the effect of initial toasting temperature (T), ellagitannin content in staves (E) and their interaction on the percentage of loss of monomeric anthocyanins.
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Table 3. Kinetic constants of degradation of monomeric anthocyanins (in months-1) in red wine aged for 12 (B and CW) and 8 months (CR) in oak barrel toasted at two initial temperatures (160 and 180 °C) and with staves of three levels of ellagitannin (LTP, MTP and HTP). Multivariate analysis of variance of the effect of initial toasting temperature (T), ellagitannin content in staves (E) and their interaction on the kinetic constants of degradation of monomeric anthocyanins.
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CW
T0
T12
T0
T8
141.5±0.8
89.6±6.4
224.9±1.1
98.6±8.5
107.1±1.1 54.3±5.3
65.4±2.2
40.1±3.0
112.5±0.8
44.3±3.8
45.1±0.1
14.5±0.4
7.3±0.7
33.1±0.6
10.4±1.2
12.1±0.3
4.3±0.5
7.8±0.3
5.0±0.4
21.4±0.1
9.4±0.9
10.3±0.0
5.5±0.7
11.3±0.2
7.6±0.5
25.4±0.1
11.4±0.9
11.1±0.0
5.7±0.6
5.2±0.4
3.1±0.2
7.7±1.1
3.0±0.3
4.9±0.0
1.8±0.2
245.8±3.7
152.8±11.3
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425.1±1.2 177.2±15.4 190.8±1.4 93.8±9.2
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[Anthocyanins] (mg/L malvidin3-O-glucoside) Malvidin-3-Oglucoside Malvidin-3-O-(6''acetyl)-glucoside Malvidin-3-O(6''p-coumaroyl)glucoside Delphinidin-3glucoside Petunidin-3glucoside Peonidin-3glucoside Total
CR
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36.7a 33.5a 38.6a 38.0a 29.8a 33.7a NS NS NS 56.6b 63.2a 54.3b 53.6b 53.3b 54.2b NS ** NS 50.9a 51.9a 40.4b 46.4ab 41.9ab 46.9ab NS NS NS
35.4a 34.2ab 35.4a 36.9a 27.5b 30.7ab NS * NS 54.9b 62.2a 52.5bc 55.0b 52.0c 53.6bc *** *** ** 52.7ab 55.8a 43.6c 47.3bc 46.9bc 48.9bc NS ** NS
40.0a 42.6a 43.5a 42.9a 36.0a 39.5a NS NS NS 59.0bc 68.4a 56.2c 61.2b 59.1bc 61.0b *** * NS 65.8ab 68.1a 56.7c 61.1bc 60.4bc 62.4abc NS ** NS
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50.9a 51.8a 51.6a 53.5a 42.7b 47.8ab NS * NS 68.3b 74.8a 66.0b 68.6b 65.8b 67.6b ** ** NS 67.0ab 69.5a 59.6c 62.5bc 61.9c 63.4bc NS ** NS
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40.1a 40.3a 40.6a 42.0a 32.8b 36.1ab NS * NS 60.8b 67.2a 58.6bc 60.4b 57.8c 59.1bc *** *** * 54.1ab 57.3a 46.2c 49.1bc 48.8bc 50.6bc NS ** NS
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37.6ab 38.5a 38.4a 40.0a 30.8b 34.6ab NS * NS 56.5b 63.3a 54.7bcd 56.3bc 52.4d 53.8cd *** *** ** 52.2ab 56.0a 43.8c 47.5bc 47.3bc 49.1bc NS ** NS
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LTP-160 LTP-180 MTP-160 MTP-180 HTP-160 B HTP-180 Toasting Ellagitannin T*E LTP-160 LTP-180 MTP-160 MTP-180 HTP-160 CR HTP-180 Toasting Ellagitannin T*E LTP-160 LTP-180 MTP-160 MTP-180 CW HTP-160 HTP-180 Toasting Ellagitannin T*E
Malvidin- Malvidin- Malvidin-3Petunidin- PeonidinDelphinidin3-O-(6''O-(6''p3-O33acetyl)coumaroyl)- 3-glucoside glucoside glucoside glucoside glucoside glucoside
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Loss (%)
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Significant letters correspond to the significant differences for each winery and each monomeric anthocyanin. NS, no significant difference; * denotes significant differences at 95% of confidence level; **denotes significant differences at 99% of confidence level; ***denotes significant differences at 99.9% of confidence level.
Total 39.0a 39.5a 39.8a 41.2a 31.9b 35.7ab NS * NS 58.5b 65.3a 56.5bc 58.2b 55.0c 56.4bc *** *** * 53.9ab 57.2a 45.5c 49.1bc 48.6bc 50.6bc NS ** NS
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CW
Toasting Ellagitannin T*E LTP-160 LTP-180 MTP-160 MTP-180 HTP-160 HTP-180
NS ** NS 0.100bc 0.120a 0.096cd 0.103b 0.091d 0.095cd
NS ** NS 0.113b 0.134a 0.107c 0.114b 0.104c 0.109bc
NS ** NS 0.140bc 0.166a 0.134c 0.144b 0.132c 0.139bc
Toasting Ellagitannin T*E LTP-160 LTP-180 MTP-160 MTP-180 HTP-160 HTP-180
*** *** ** 0.060b 0.068a 0.049d 0.054bcd 0.053cd 0.056bc
*** *** ** 0.067b 0.074a 0.055d 0.060cd 0.058cd 0.062bc
Toasting Ellagitannin T*E
** *** NS
** *** NS
Petunidin3glucoside
Peonidin3glucoside
Total
0.037ab 0.035ab 0.040a 0.040a 0.031b 0.032ab
0.039ab 0.036abc 0.039ab 0.042a 0.029c 0.032bc
0.053a 0.050ab 0.051ab 0.055a 0.043b 0.047ab
0.044ab 0.043ab 0.044ab 0.047a 0.034c 0.039bc
NS * NS 0.091b 0.113a 0.085bc 0.090bc 0.082c 0.086bc
NS ** NS 0.098bc 0.118a 0.093cd 0.100b 0.090d 0.095bcd
NS NS NS 0.121bc 0.149a 0.116c 0.129b 0.119c 0.124bc
NS ** NS 0.106bc 0.127a 0.101cd 0.108b 0.097d 0.102cd
*** *** * 0.095ab 0.102a 0.080c 0.086c 0.083c 0.088bc
*** *** ** 0.063ab 0.070a 0.048c 0.059bc 0.050c 0.058bc
*** *** ** 0.064b 0.072a 0.051d 0.057cd 0.055cd 0.059bc
*** *** ** 0.092b 0.101a 0.080c 0.084c 0.083c 0.088bc
*** *** *** 0.065b 0.072a 0.053d 0.059bcd 0.056cd 0.060bc
* *** NS
** ** NS
** *** NS
* *** NS
** *** NS
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Delphinidin3-glucoside
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Malvidin-3O-(6''pcoumaroyl)glucoside 0.063ab 0.062ab 0.064ab 0.070a 0.049c 0.057bc
Malvidin3-Oglucoside
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0.042ab 0.041ab 0.042ab 0.045a 0.032c 0.037bc
Malvidin-3O-(6''acetyl)glucoside 0.046ab 0.044ab 0.046ab 0.049a 0.036c 0.039bc
k (months-1)
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Significant letters correspond to the significant differences for each winery and each monomeric anthocyanin. NS, no significant difference; * denotes significant differences at 95% of confidence level; **denotes significant differences at 99% of confidence level; ***denotes significant differences at 99.9% of confidence level.
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Figure captions.
Figure 1. Scheme of the experiments, including oak barrel conditions and number of replicates.
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Figure 2. Evolution of ellagic acid (mg/L) in CW red wines aged in oak barrel for 12 months. The ellagic acid concentration in B and CR wines at each sampling time is available in a previously published article (Watrelot et al., 2018). Figure 3. Percentage of pigmented tannins in CW red wines after 12 months of aging in oak barrels. These values in B and CR wines can be found in Watrelot et al., (2018).
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Figure 4. Color intensity (A) and hue (B) of red wines (B, CR and CW) aged for 8 to 12 months in oak barrels.
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Figure 1.
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Figure 2.
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LTP, low ellagitannin level; MTP, medium ellagitannin level; HTP, high ellagitannin level. 160, toasted at 160 °C as initial temperature; 180, toasted at 180 °C as initial temperature. T0 corresponds to the ellagic acid concentration in red wine prior to barreling. Different letters in the bars correspond to significant differences between oak barrel modalities for each aging time.
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Figure 3.
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LTP, low ellagitannin level; MTP, medium ellagitannin level; HTP, high ellagitannin level. 160, toasted at 160 °C as initial temperature; 180, toasted at 180 °C as initial temperature. Different letters in the bars correspond to significant differences between the oak barrel modalities.
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Figure 4.
LTP, low ellagitannin level; MTP, medium ellagitannin level; HTP, high ellagitannin level. 160, toasted at 160 °C as initial temperature; 180, toasted at 180 °C as initial temperature. Different letters in the bars correspond to significant differences between oak barrel modalities for each winery at a given time.
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Highlights Malvidin coumaroyl glucoside is the most reactive anthocyanin during barrel aging
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Low ellagitannin barrels accelerate the formation of stable wine pigments
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Oak barrel modalities did not impact on the wine color intensity at one year
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S0023-6438(18)30757-6
DOI:
10.1016/j.lwt.2018.09.025
Reference:
YFSTL 7405
To appear in:
LWT - Food Science and Technology
Received Date: 31 May 2018 Revised Date:
29 August 2018
Accepted Date: 11 September 2018
Please cite this article as: Watrelot, A.A., Waterhouse, A.L., Oak barrel tannin and toasting temperature: Effects on red wine anthocyanin chemistry, LWT - Food Science and Technology (2018), doi: https:// doi.org/10.1016/j.lwt.2018.09.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Oak barrel tannin and toasting temperature: Effects on red wine anthocyanin chemistry.
3 Aude A. Watrelot*, Andrew L. Waterhouse
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Department of Viticulture and Enology, University of California Davis, One Shields Ave., Davis, CA 95616-5270, USA
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*corresponding author:
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A.A. Watrelot
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Department of Viticulture and Enology, University of California Davis, One Shields
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Ave., Davis, CA 95616-5270, USA
Telephone: +1-530-752-5054
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Fax: +1-530-752-0382 E-mail: [email protected]; [email protected]
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Abstract Oak-derived compounds are generally considered to be important in red wine
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perception and color qualities. In this study, the percentage of loss and kinetic rates of
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degradation of monomeric anthocyanins as well as the formation of pigmented tannins were
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monitored in red wines aged for 8 and 12 months in oak barrels differentiated by stave
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ellagitannin content (low LTP, medium MTP and high HTP) and toasting level (160 and 180
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°C as initial temperature). The malvidin coumaroyl glucoside is found to be the most reactive
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monomeric anthocyanin during barrel aging. While color intensity was not affected, the
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results indicated that in presence of high ellagitannin content red wines exhibit a lower loss
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and slower degradation of monomeric anthocyanins and slower formation of pigmented
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tannins.
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Keywords Monomeric
anthocyanins,
kinetic
rates,
wood
toasting
level,
ellagitannin
concentration, pigmented tannins
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1. Introduction
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Anthocyanins are the most important pigments (of the Greek anthos = flower and kianos =
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blue) of the vascular plants, present in vacuoles. These pigments are water-soluble and
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responsible for pink to blue color range in flowers and fruits. The structure of anthocyanidins
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ACCEPTED MANUSCRIPT (anthocyanin aglycones) is a 2-phenyl-1-benzopyrylium nucleus diversely substituted by OH
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and OMe groups, especially on the B-ring. The pigment color depends on several factors,
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such as substitution on the B-ring, pH, presence of copigments and metal ions (Dangles,
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Stoeckel, Wigand, & Brouillard, 1992). Anthocyanins found in red wine (mostly malvidin-3-
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O-glucoside) are extracted from grapes during winemaking process and are able to interact
46
with cell wall material (Buchweitz, Nagel, Carle, & Kammerer, 2012) but also with
47
acetaldehyde, pyruvic acid, condensed tannins and hydrolysable tannins to form new products
48
such as vitisin A and B, anthocyanin-flavanol, and anthocyanin-ellagitannin allowing color
49
stabilization (Chassaing et al., 2010; Mateus & de Freitas, 2001).
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Ellagitannins are hydrolysable tannins from wood that are easily extracted by wine that is
51
a hydroalcoholic solution. Due to their chemical structure they are known to be the first
52
barrier against phenolic compounds oxidation but ellagitannins can be degraded during the
53
barrel processing, especially toasting. Higher toasting temperatures and the duration of the
54
toasting process lead to higher degradation of ellagitannins, (Chira & Teissedre, 2015;
55
Fernández de Simón, Cadahía, del Álamo, & Nevares, 2010). Many studies have focused on
56
the color properties of red wine aged in oak barrels or in stainless tanks with oak chips and on
57
the formation of new pigments involving anthocyanins that could explain the color
58
stabilization (De Rosso, Panighel, Dalla Vedova, Stella, & Flamini, 2009; Dumitriu, Lerma,
59
Cotea, Zamfir, & Peinado, 2016; Ortega-Heras, Pérez-Magariño, Cano-Mozo, & González-
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San José, 2010). The addition of wood chips during wine aging induced a higher stabilization
61
of color and various level of degradation of monomeric anthocyanins depending on the type
62
of wood, due to their porosity that lead to different rate of ellagitannins extraction (Gambuti,
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Capuano, Lisanti, Strollo, & Moio, 2010; Kyraleou et al., 2016). However few investigations
64
have addressed the effect of oak barrel ellagitannin levels or toasting temperature on the fate
65
of anthocyanins (Mateus & de Freitas, 2001). Moreover, the use of enological tannins in red
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tannins are not well characterized and their effects are controversial. In many articles,
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enological tannins contribute to red wine color stabilization and mouthfeel perception
69
(Bautista-Ortin, Cano-Lechuga, Ruiz-Garcia, & Gomez-Plaza, 2014; Obreque-Slier, Peña-
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Neira, & López-Solís, 2012), however other studies show that the addition of enological
71
tannins to red wine does not have a significant effect and/or has a negative effect on red wine
72
quality (Harbertson, Parpinello, Heymann, & Downey, 2012). In our previous article
73
(Watrelot et al., 2018), the effect of oak barrel tannin and toasting temperature on condensed
74
tannin chemistry has been elucidated and some data such as ellagic acid content and the
75
percentage of pigmented tannins has been discussed . The aim of this work is to complement
76
the previous one, investigating the effect of the level of oak barrel tannin and toasting
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temperature on the degradation rates of wine anthocyanins and on the formation of pigmented
78
tannins.
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2. Material and Methods
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2.1. Reagent and chemicals
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All solvents were HPLC grade. Acetonitrile, methanol, acetic acid, L- (+) ascorbic
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acid, o-phosphoric acid and ammonium dihydrogen phosphate were purchased from VWR
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International (Radnor, PA). (-)-epicatechin, (+)-catechin hydrate and ellagic acid were
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purchased from Sigma-Aldrich (St. Louis, MO). Oenin chloride was purchased from
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Extrasynthèse (Lyon, France).
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2.2. Oak barrel characteristics
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The barrels (225 L) were made from oak (Q. sessilis) coming from French forests (90
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90
coopering.
91
2.2.1. Oak wood sorting methodology according to its tannin potential (TP)
92
Wood classification according to its ellagitannin content was performed by near-infrared
93
(NIR) spectroscopy using a technique based on the use of an acousto-optic tunable filter
94
(AOTF, Brimrose, USA) (Watrelot et al., 2018). Briefly, after machining, the untoasted staves
95
were first being intended to gather spectral data by NIR and then ellagitannins total level
96
analyzed through ellagic acid dosage in HPLC-DAD after extraction and hydrolysis in acidic
97
medium. The calibration was performed from a partial least squares (PLS) regression after
98
selecting the most discriminant spectral zones. The correlation coefficient between spectral
99
measurement and HPLC dosage of total ellagitannins (0.89) shows the performance of the
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model used. The calibration has been carried out on 450 samples from 27 different groups.
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The classification from the NIR method was used to sort the staves prior to toasting into three
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groups of tannin potential (TP) i.e. the ellagitannin content in untoasted wood: Low or LTP <
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4000, Medium or MTP from 4001 to 6000 and High or HTP from 6001 to 8000 µg of ellagic
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acid equivalent / g of dry wood.
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2.2.2. Wood toasting methodology
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Toasting was conducted after automated steam bending (4 min), which yields a neutral
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untoasted barrel. Toasting, controlled by computer, was performed using radiant heat rather
108
than direct contact with flame. The toasting pot was fed regularly with fuel (100 % oak
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pellets) by an auger. The barrel, placed on a turntable, rotates around a double cone that
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covers the fire and channels the heat source, during the entire toasting phase. An infrared
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sensor, performing measurements on the internal surface of the shell, provides temperature
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control, with a heating accuracy of +/- 3 ° C. In our study, all barrels (LTP, MTP and HTP)
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initial temperatures were compared (160 and 180 °C), as previously described by Watrelot et
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al., 2018.
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2.3. Red wine winemaking
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Red wines were made by two Californian wineries (B and CR) and one winery from
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Washington State (CW) from 2015 Cabernet Sauvignon grapes of Napa Valley and Wahluke
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slope, respectively. The winemaking process of B and CR is already described in Watrelot et
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al., 2018. Briefly, the alcoholic fermentation was made using Zymaflore F15 and D254 yeast
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strains in B and CR wines, respectively. Spontaneous malolactic fermentations occurred after
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yeast fermentation was complete. Sulfur dioxide, 60 mg/L, was added to the wines prior to
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barreling and three wine samples of each winery were analyzed. The CW wine was made
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from grapes harvested by the beginning of October 2015. The wine was fermented for 5 days
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at 26.7 °C with Red Start Premier Cuvee yeast strain. Following inoculation with Oenococcus
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oeni, the malolactic fermentation of CW wine in tank took 10 days prior to barreling. 60 mg/L
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of oak flour (Innerstave Oak plus flour) was added into the blend prior to barreling. Each wine
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was barreled in 18 barrels (225 L) (three barrels for each 6 modalities previously described).
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Barrels were topped every month, racked every three months and wine samples were analyzed
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after each racking time. B and CW wines were aged for 12 months in 18 barrels (triplicate of
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the 6 barrel modalities previously described) and CR wines were aged for 8 months in 18
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barrels (triplicate of the 6 barrel modalities previously described) as decided by the winery
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involved in this study (Figure 1).
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2.4. Red wine characterization
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2.4.1. Sampling
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For each winery, three wine samples were collected before barreling (named T0) and 6
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160, LTP-180, MTP-160, MTP-180, HTP-160 and HTP-180) and aged for 3, 5, 8 and 12
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months for wine from B and CW winery. After 3 months of aging, three wine samples were
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collected from each oak barrel modalities (named T3), corresponding to 18 samples for each
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winery. The same sampling was carried out after 5, 8 and 12 months of aging (T5, T8 and
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T12). CR wines were aged for 8 months and the sampling was carried out on wines after 3, 5
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and 8 months of aging. To summarize, a total of 18 wine samples per winery at each aging
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time was analyzed (Figure 1). pH, ethanol content and titratable acidity were determined
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using a Winescan FT120 (FOSS, Eden Prairie, Minnesota, USA) for each modality and
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replicate and the average of the eighteen wines was used to compare these parameters
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between wineries. At the end of aging, the pH was 3.6, 3.7 and 3.8 in CR, B and CW wines,
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respectively; the ethanol content was 14.9, 15.2 and 14.7 % vol in CR, B and CW wines,
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respectively and the titratable acidity was 6.0, 6.0 and 5.3 g/L in CR, B and CW wines,
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respectively.
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2.4.2. Ellagic acid concentration
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The total ellagitannin concentration of red wine was determined by the quantification
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of ellagic acid released after acidic hydrolysis and HPLC quantification, as previously
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described (Watrelot et al., 2018). In this study, the ellagic acid concentration of CW wines at
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each sampling time and of B and CR wines only at the end of aging are shown. The ellagic
156
acid concentration in B and CR wines at each sampling time is available in a previously
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published article (Watrelot et al., 2018).
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2.4.3. Monomeric anthocyanins
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Monomeric anthocyanins of red wines were characterized by C18 reversed-phase
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HPLC-DAD after direct injection of 20µL filtered wine (0.45µm, PTFE filters) according to 7
ACCEPTED MANUSCRIPT (Ritchey & Waterhouse, 1999). The 1100 Agilent HPLC system was composed of a degasser,
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an autosampler, a quaternary pump and a diode array detector, controlled by Chemstation
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software. For analysis, a reverse phase 250 × 4 mm, 5µm, RP-18 Lichrospher® 100 column
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was used. The mobile phases used were composed of solvent A (50 mM ammonium
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dihydrogen phosphate pH 2.6), solvent B (20% solvent A in acetonitrile) and solvent C (0.2
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M ortho-phosphoric acid in water, pH1.5) with the following elution gradient: 0 min (0 %B; 0
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%C), 8 min (8 %B; 0 %C), 20 min (14 %B; 86 %C), 25 min (16.5 %B; 82 %C), 35 min (21.5
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%B; 78.5 %C), 70 min (50 %B; 50 %C), 75 min (0 %B; 0 %C), 90 min (0 %B; 0 %C). The
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flow rate was set at 0.5 mL/min and the column temperature at 40 °C. Detection was made at
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520 nm. Monomeric anthocyanin concentration is expressed in malvidin-3-O-glucoside
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equivalents.
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2.4.4. Pigmented tannins, color intensity and hue
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The percentage of pigmented tannins was measured by gel permeation chromatography and
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corresponded to the ratio of absorbance at 520 nm to 280 nm, as previously described
175
(Watrelot et al., 2018). In this study, only the percentage of pigmented tannins in CW wines is
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presented. These values in B and CR wines can be found in Watrelot et al. (2018). The
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spectrum of all red wines at the end of aging was determined using 10 mm polymethyl
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methacrylate (PMMA) cuvettes by a UV-Visible spectrophotometer. The absorbance values at
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420 nm, 520 nm and 620 nm were analyzed to determine the color intensity (sum of the
180
absorbances at these three wavelengths) and the hue (420 nm: 520 nm ratio) of red wines for
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the three wineries and all oak barrel modalities. The analyses were done for each wine
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(experimental triplicates).
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2.5. Statistical analysis Experiments were performed in triplicate: red wine was aged in three barrels for each 8
ACCEPTED MANUSCRIPT modality and analysis was performed on each wine (18 per winery), so the results are
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expressed as the mean and the standard deviation of experimental replicates and all statistical
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analysis were made using XLstat software for Microsoft Excel®. One way-ANOVA using a
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Fisher (LSD) test with a confidence interval of 95 % was carried out on all samples to identify
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significant differences between all barrel modalities for each winery. A multivariate analysis
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of variance (MANOVA) using toasting temperature and ellagitannin content as factors was
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performed to determine the effect of these individual factors and their interactions on the
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monomeric anthocyanin loss percentage and their degradation rates for all three wineries.
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3. Results and Discussion
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3.1. Ellagic acid concentration
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Ellagitannins from wood are extractable compounds in wine due to the hydroalcoholic
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medium. As previously observed in B and CR wines (Watrelot et al., 2018), the extraction of
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ellagitannin into CW red wine increased with aging (Figure 2), even though this wine had
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some ellagitannins added prior to barreling (T0). The increase of ellagic acid concentrations
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in the red wines between oak barrel modalities were the same up to 8 months of aging, as
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previously observed, and were not significantly different at the end of aging (12 months). This
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is due to the slow extraction for a few months after wine contact and to an equilibrium that
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probably occur between ellagitannins already and still extracted (Michel et al., 2011). After 8
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months of aging, ellagic acid concentration was the highest in red wine aged in oak barrel rich
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in ellagitannin and toasted at low temperature (HTP-160) (Figure 2) and was the lowest in red
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wine aged in oak barrel with low ellagitannin content and toasted at higher temperature (LTP-
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180). In accordance with Michel, Jourdes, Giordanengo, Mourey, & Teissedre, (2012), staves
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rich in ellagitannin (HTP) release higher ellagitannin content in wine. Higher ellagitannin
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ellagitannins and other wood compounds are degraded by high temperature (Chira &
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Teissedre, 2015).
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3.2. Monomeric anthocyanins degradation of red wine aged in oak barrels
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Large variation of monomeric anthocyanin concentrations was observed between the
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three wines aged in oak barrels (Table 1). As expected, malvidin-3-O-glucoside was the most
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abundant followed by the acetylated form of malvidin-3-O-glucoside. Prior to barreling (T0),
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CR wines were richer in total monomeric anthocyanin (425.1 mg/L as malvidin-3-O-
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glucoside eq.) than B and CW wines (245.8 and 190.8 mg/L as malvidin-3-O-glucoside eq.,
218
respectively) which can be related to the initial anthocyanin concentration in grapes and to the
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winemaking process. The concentrations of each monomeric anthocyanin are in the typical
220
range of concentrations found in red wines from Cabernet sauvignon grapes (Monagas,
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Gómez-Cordovés, & Bartolomé, 2005). At the end of barrel aging, the concentration of each
222
monomeric anthocyanin in B and CR wines was similar, except delphinidin-3-glucoside and
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petunidin-3-glucoside were slightly higher in CR wines.
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The percentages of loss of monomeric anthocyanins during red wine aging in the oak
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barrels toasted at two temperatures and of three levels of ellagitannin are shown in Table 2. In
226
accordance with the concentrations of anthocyanins prior to barreling and at the end of aging
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(Table 1), the percentage of loss of total monomeric anthocyanin was higher in CR wines than
228
CW and B wines. This indicated that the anthocyanin content of the CR wines was less stable
229
during aging. This wine had the highest concentration of condensed tannins in the wine at the
230
end of aging (943 mg/L as catechin equivalent (Watrelot et al., 2018)), and we expect these
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tannins react with anthocyanin, leading to the loss of monomeric anthocyanins, as observed
232
by others (García-Estévez et al., 2013). In spite of having the highest concentration in red
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234
degraded during aging. Whatever the aging modalities, the percentage of loss of monomeric
235
anthocyanin followed this range: Malvidin-3-O-(6''p-coumaroyl)-glucoside > peonidin-3-
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glucoside > malvidin-3-O-(6''-acetyl)-glucoside > malvidin-3-O-glucoside > delphinidin-3-
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glucoside ≥ petunidin-3-glucoside, following a pattern observed in Port wines (Mateus & de
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Freitas, 2001) as well as in Cabernet Sauvignon and Tempranillo wines (Monagas et al.,
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2005). The percentage of loss of malvidin-3-O-glucoside, delphinidin-3-glucoside and
240
petunidin-3-glucoside was very similar (significantly identified but not shown) suggesting
241
that the number of hydroxylation or methoxylation of the three groups on the B-ring does not
242
play a major role in their degradation, as observed in blueberry juice (Howard, Brownmiller,
243
Mauromoustakos, & Prior, 2016). However, the percentage of loss of peonidin-3-glucoside
244
that is an anthocyanin with 1 hydroxyl group and 1 methoxyl group on the B-ring was much
245
higher than the anthocyanin with 3 groups on the B-ring, suggesting that the number of
246
groups on the B-ring is involved in the stability of the monomeric anthocyanins (Castañeda-
247
Ovando, Pacheco-Hernández, Páez-Hernández, Rodríguez, & Galán-Vidal, 2009). As the
248
level of glycosylation of the anthocyanin, the type of acetylation showed an effect on the loss
249
of anthocyanin, for which the coumaroyl malvidin was more degraded than the acetylated
250
malvidin (Mateus & de Freitas, 2001; Monagas et al., 2005). It is possible that the loss of the
251
acylated anthocyanins was due to ester hydrolysis of the acyl group or to precipitation of it
252
(Gil et al., 2017) and not oxidation of the anthocyanidin. The effect of the oak barrel
253
modalities on the loss of monomeric anthocyanins varied depending on the red wine. B and
254
CW wines did not show any significant effect of the toasting temperature (Table 2) in contrast
255
to CR wines where 180 °C significantly increased the percentage of loss of all monomeric
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anthocyanins except delphidin-3-glucoside, compared to 160 °C. This effect was significant
257
only in CR wines probably due to the higher total monomeric anthocyanin concentration
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259
concentration in red wines, that can limit their barrier effect against oxidation, leading
260
therefore to the loss of anthocyanins. This agrees with Gonzalez-Saiz et al. (2014) who
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showed that the content of anthocyanins decreased in wine that infused with medium to
262
medium-plus toasted wood chips.
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For all wines, the level of ellagitannin in staves showed significant effect on the loss of
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anthocyanins (Figure 2 and Table 2). Wine aged in low ellagitannin content (LTP) barrels
265
showed a higher percentage of loss than in medium and high ellagitannin content, and
266
therefore contained significantly less monomeric anthocyanins at 12 months. This showed
267
once again that ellagitannins in wine from barrel are able to protect the anthocyanins from
268
their degradation by oxidation during aging, and not necessary interact with them to form new
269
stable pigment (Chassaing et al., 2010). The combined effect of toasting and ellagitannin
270
content in staves was significant only in CR wines for malvidin-3-O-glucoside, malvidin-3-O-
271
(6''-acetyl)-glucoside and the petunidin-3-glucoside (Table 2). Wine aged in LTP-180 barrels
272
that contained the least ellagitannins induced the highest loss of anthocyanins in contrast to
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HTP-160 that contained the highest ellagitannin content.
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In agreement with some studies (Bakker, 2015; Mateus & de Freitas, 2001; Oliveira,
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Barros, Silva Ferreira, & Silva, 2015), the degradation reaction of anthocyanins follows first-
276
order kinetics. The rate constants (k) were calculated from the slope after linear regression of
277
ln (C/C0) = f(t), where C is the concentration of anthocyanin, C0 is the concentration of
278
anthocyanin prior barreling and t is the aging time in months (Figure 1 in supplementary
279
information). The rate constant varied depending on the anthocyanin structure as well as on
280
the oak barrel modality (Table 3). Like the percentage of loss, the rate constant of degradation
281
was higher for malvidin-3-O-(6''p-coumaroyl)-glucoside and was the lowest for delphinidin-
282
3-glucoside, for all three red wines (B, CR and CW). The degradation rate constant of
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malvidin-3-O-glucoside was lower than those of acylated malvidin derivatives (malvidin-3-O-
284
6’’-acetyl-glucoside and malvidin-3-O-6’’-coumaroyl-glucoside), in accord with Monagas et
285
al., (2005) and Mateus & de Freitas, (2001). As noted above, the loss of acylated
286
anthocyanins likely involves ester hydrolysis as well as oxidation.
287
components are fairly small, the products, the simple glycosides, would not increase by
288
noticeable amount. Between the three wines, the rate constant of total anthocyanin loss varied
289
from 0.041 to 0.101 months-1 in B and CR, respectively. B and CW wines showed similar rate
290
constants even if B had the lowest values, while in CR wines the rate constants were much
291
higher. The kinetic rate constants were significantly higher in CR and CW wines aged in oak
292
barrel toasted at 180 °C than at 160 °C (Table 3), revealing that the toasting temperature of
293
oak barrel affects the kinetics of degradation of monomeric anthocyanins. As explained
294
above, the higher thermolytic degradation of ellagitannins from wood during the toasting
295
process at 180 °C (initial toasting temperature) (Chira & Teissedre, 2015) reduces their
296
protective effect against anthocyanin degradation.
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In the three wines, the rate constants for all anthocyanins were significantly affected
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by the ellagitannin content in staves, where higher k values were observed in wines aged in
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LTP barrels. This confirms the above observation where low ellagitannin content (due to
300
thermolytic degradation or due to low initial concentration in wood) induced a faster loss of
301
monomeric anthocyanins due to the lack of compounds protecting against oxidation reactions.
302
Wine aged in LTP-180 barrels showed a faster loss (higher k value) compared to wine aged in
303
HTP-160 (which contained the highest concentration of ellagitannins). The loss of monomeric
304
anthocyanins is usually associated with the formation of anthocyanin-pyruvic acid adducts
305
(vitisin A) (Monagas et al., 2005), but this compound was not found in our samples probably
306
because the sensitivity of detection of our method was too low.
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As reported earlier, the percentage of pigmented tannins at the end of aging was 13
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309
respectively), which might be related to the highest loss percentage and the fastest
310
degradation of monomeric anthocyanin in wines from CR winery. The highest concentration
311
of pigmented tannins (Figure 3 and Watrelot et al., 2018) was observed in wine aged in LTP-
312
180 and the lowest in HTP-160, suggesting that in presence of a low concentration of
313
ellagitannins, the monomeric anthocyanins are more able to interact with condensed tannins to
314
form pigmented tannins. As suggested by Gambuti et al., (2017), the oxidation occurring
315
during red wine aging responsible for tannin chemical reactions is a key parameter in the
316
formation of pigmented tannins, but also the phenolic structure. The tannins: anthocyanins (T:
317
A) ratio might be an indicator of the formation of pigmented tannins that is supposed to
318
correlate with the formation of pigmented tannins identified above. In our study, the T: A
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ratio, based on tannin and anthocyanin concentrations at the end of aging, was higher in LTP-
320
180 wines from CR and CW wines, while for all wines, the T: A ratio was lower in HTP-160
321
wines (data not shown), which was in agreement with the formation of pigmented tannins
322
observed. As previously reported by Boulton, (2001), conversion of monomeric anthocyanins
323
to pigmented tannins during aging might be responsible for stable red wine color. In order to
324
assess this hypothesis, the color intensity and hue of all red wines at the end of aging were
325
measured, based on their UV-Vis spectra (Figure 4). No clear significant effect was observed
326
in term of color intensity (Figure 4A) and hue (Figure 4B) for all wines, regarding oak barrel
327
modalities. In B wines, the color intensity seemed to be higher in LTP-180 wines, but this was
328
not observed in other wineries. As previously explained by Harbertson, et al., (2012) the
329
addition of enological tannins to wine does not always provide an improvement of the
330
mouthfeel or color. In this study, the lack of influence of oak barrel ellagitannin levels on red
331
wine color might be attributed to the loss of anthocyanins combined with the formation of
332
new pigments.
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334
observed, oak barrels rich in ellagitannins led to a slower and lesser loss of monomeric
335
anthocyanins. These barrels were also associated with a lesser formation of pigmented
336
tannins.
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The authors thank the three wineries from California and Washington State involved in
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4. Acknowledgments
this project for providing red wines.
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This study was funded by Vicard Generation 7, France.
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References
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Bakker, J. (2015). HPLC of anthocyanins in port wines: Determination of ageing rates. VITIS
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- Journal of Grapevine Research, 25(4), 203. Bautista-Ortin, A. B., Cano-Lechuga, M., Ruiz-Garcia, Y., & Gomez-Plaza, E. (2014).
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Interactions between grape skin cell wall material and commercial enological tannins.
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Practical implications. Food Chemistry, 152, 558‑565.
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https://doi.org/10.1016/j.foodchem.2013.12.009
Boulton, R. (2001). The Copigmentation of Anthocyanins and Its Role in the Color of Red Wine: A Critical Review. American Journal of Enology and Viticulture, 52(2), 67‑87.
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Buchweitz, M., Nagel, A., Carle, R., & Kammerer, D. R. (2012). Characterisation of sugar
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beet pectin fractions providing enhanced stability of anthocyanin-based natural blue
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food colourants. Food Chemistry, 132(4), 1971‑1979.
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Table captions. Table 1. Concentrations of monomeric anthocyanins (expressed in mg/L malvidin-3-Oglucoside) in red wine prior to barreling (T0) and after aging in oak barrels (B, CR, CW wines).
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Table 2. Percentage of loss of monomeric anthocyanins in red wine aged for 12 (B and CW) and 8 months (CR) in oak barrel toasted at two initial temperatures (160 and 180 °C) and with staves of three levels of ellagitannin (LTP, MTP and HTP). Multivariate analysis of variance of the effect of initial toasting temperature (T), ellagitannin content in staves (E) and their interaction on the percentage of loss of monomeric anthocyanins.
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Table 3. Kinetic constants of degradation of monomeric anthocyanins (in months-1) in red wine aged for 12 (B and CW) and 8 months (CR) in oak barrel toasted at two initial temperatures (160 and 180 °C) and with staves of three levels of ellagitannin (LTP, MTP and HTP). Multivariate analysis of variance of the effect of initial toasting temperature (T), ellagitannin content in staves (E) and their interaction on the kinetic constants of degradation of monomeric anthocyanins.
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CW
T0
T12
T0
T8
141.5±0.8
89.6±6.4
224.9±1.1
98.6±8.5
107.1±1.1 54.3±5.3
65.4±2.2
40.1±3.0
112.5±0.8
44.3±3.8
45.1±0.1
14.5±0.4
7.3±0.7
33.1±0.6
10.4±1.2
12.1±0.3
4.3±0.5
7.8±0.3
5.0±0.4
21.4±0.1
9.4±0.9
10.3±0.0
5.5±0.7
11.3±0.2
7.6±0.5
25.4±0.1
11.4±0.9
11.1±0.0
5.7±0.6
5.2±0.4
3.1±0.2
7.7±1.1
3.0±0.3
4.9±0.0
1.8±0.2
245.8±3.7
152.8±11.3
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425.1±1.2 177.2±15.4 190.8±1.4 93.8±9.2
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[Anthocyanins] (mg/L malvidin3-O-glucoside) Malvidin-3-Oglucoside Malvidin-3-O-(6''acetyl)-glucoside Malvidin-3-O(6''p-coumaroyl)glucoside Delphinidin-3glucoside Petunidin-3glucoside Peonidin-3glucoside Total
CR
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36.7a 33.5a 38.6a 38.0a 29.8a 33.7a NS NS NS 56.6b 63.2a 54.3b 53.6b 53.3b 54.2b NS ** NS 50.9a 51.9a 40.4b 46.4ab 41.9ab 46.9ab NS NS NS
35.4a 34.2ab 35.4a 36.9a 27.5b 30.7ab NS * NS 54.9b 62.2a 52.5bc 55.0b 52.0c 53.6bc *** *** ** 52.7ab 55.8a 43.6c 47.3bc 46.9bc 48.9bc NS ** NS
40.0a 42.6a 43.5a 42.9a 36.0a 39.5a NS NS NS 59.0bc 68.4a 56.2c 61.2b 59.1bc 61.0b *** * NS 65.8ab 68.1a 56.7c 61.1bc 60.4bc 62.4abc NS ** NS
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50.9a 51.8a 51.6a 53.5a 42.7b 47.8ab NS * NS 68.3b 74.8a 66.0b 68.6b 65.8b 67.6b ** ** NS 67.0ab 69.5a 59.6c 62.5bc 61.9c 63.4bc NS ** NS
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40.1a 40.3a 40.6a 42.0a 32.8b 36.1ab NS * NS 60.8b 67.2a 58.6bc 60.4b 57.8c 59.1bc *** *** * 54.1ab 57.3a 46.2c 49.1bc 48.8bc 50.6bc NS ** NS
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37.6ab 38.5a 38.4a 40.0a 30.8b 34.6ab NS * NS 56.5b 63.3a 54.7bcd 56.3bc 52.4d 53.8cd *** *** ** 52.2ab 56.0a 43.8c 47.5bc 47.3bc 49.1bc NS ** NS
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LTP-160 LTP-180 MTP-160 MTP-180 HTP-160 B HTP-180 Toasting Ellagitannin T*E LTP-160 LTP-180 MTP-160 MTP-180 HTP-160 CR HTP-180 Toasting Ellagitannin T*E LTP-160 LTP-180 MTP-160 MTP-180 CW HTP-160 HTP-180 Toasting Ellagitannin T*E
Malvidin- Malvidin- Malvidin-3Petunidin- PeonidinDelphinidin3-O-(6''O-(6''p3-O33acetyl)coumaroyl)- 3-glucoside glucoside glucoside glucoside glucoside glucoside
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Loss (%)
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Significant letters correspond to the significant differences for each winery and each monomeric anthocyanin. NS, no significant difference; * denotes significant differences at 95% of confidence level; **denotes significant differences at 99% of confidence level; ***denotes significant differences at 99.9% of confidence level.
Total 39.0a 39.5a 39.8a 41.2a 31.9b 35.7ab NS * NS 58.5b 65.3a 56.5bc 58.2b 55.0c 56.4bc *** *** * 53.9ab 57.2a 45.5c 49.1bc 48.6bc 50.6bc NS ** NS
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CW
Toasting Ellagitannin T*E LTP-160 LTP-180 MTP-160 MTP-180 HTP-160 HTP-180
NS ** NS 0.100bc 0.120a 0.096cd 0.103b 0.091d 0.095cd
NS ** NS 0.113b 0.134a 0.107c 0.114b 0.104c 0.109bc
NS ** NS 0.140bc 0.166a 0.134c 0.144b 0.132c 0.139bc
Toasting Ellagitannin T*E LTP-160 LTP-180 MTP-160 MTP-180 HTP-160 HTP-180
*** *** ** 0.060b 0.068a 0.049d 0.054bcd 0.053cd 0.056bc
*** *** ** 0.067b 0.074a 0.055d 0.060cd 0.058cd 0.062bc
Toasting Ellagitannin T*E
** *** NS
** *** NS
Petunidin3glucoside
Peonidin3glucoside
Total
0.037ab 0.035ab 0.040a 0.040a 0.031b 0.032ab
0.039ab 0.036abc 0.039ab 0.042a 0.029c 0.032bc
0.053a 0.050ab 0.051ab 0.055a 0.043b 0.047ab
0.044ab 0.043ab 0.044ab 0.047a 0.034c 0.039bc
NS * NS 0.091b 0.113a 0.085bc 0.090bc 0.082c 0.086bc
NS ** NS 0.098bc 0.118a 0.093cd 0.100b 0.090d 0.095bcd
NS NS NS 0.121bc 0.149a 0.116c 0.129b 0.119c 0.124bc
NS ** NS 0.106bc 0.127a 0.101cd 0.108b 0.097d 0.102cd
*** *** * 0.095ab 0.102a 0.080c 0.086c 0.083c 0.088bc
*** *** ** 0.063ab 0.070a 0.048c 0.059bc 0.050c 0.058bc
*** *** ** 0.064b 0.072a 0.051d 0.057cd 0.055cd 0.059bc
*** *** ** 0.092b 0.101a 0.080c 0.084c 0.083c 0.088bc
*** *** *** 0.065b 0.072a 0.053d 0.059bcd 0.056cd 0.060bc
* *** NS
** ** NS
** *** NS
* *** NS
** *** NS
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Delphinidin3-glucoside
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Malvidin-3O-(6''pcoumaroyl)glucoside 0.063ab 0.062ab 0.064ab 0.070a 0.049c 0.057bc
Malvidin3-Oglucoside
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0.042ab 0.041ab 0.042ab 0.045a 0.032c 0.037bc
Malvidin-3O-(6''acetyl)glucoside 0.046ab 0.044ab 0.046ab 0.049a 0.036c 0.039bc
k (months-1)
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Significant letters correspond to the significant differences for each winery and each monomeric anthocyanin. NS, no significant difference; * denotes significant differences at 95% of confidence level; **denotes significant differences at 99% of confidence level; ***denotes significant differences at 99.9% of confidence level.
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Figure captions.
Figure 1. Scheme of the experiments, including oak barrel conditions and number of replicates.
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Figure 2. Evolution of ellagic acid (mg/L) in CW red wines aged in oak barrel for 12 months. The ellagic acid concentration in B and CR wines at each sampling time is available in a previously published article (Watrelot et al., 2018). Figure 3. Percentage of pigmented tannins in CW red wines after 12 months of aging in oak barrels. These values in B and CR wines can be found in Watrelot et al., (2018).
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Figure 4. Color intensity (A) and hue (B) of red wines (B, CR and CW) aged for 8 to 12 months in oak barrels.
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Figure 1.
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Figure 2.
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LTP, low ellagitannin level; MTP, medium ellagitannin level; HTP, high ellagitannin level. 160, toasted at 160 °C as initial temperature; 180, toasted at 180 °C as initial temperature. T0 corresponds to the ellagic acid concentration in red wine prior to barreling. Different letters in the bars correspond to significant differences between oak barrel modalities for each aging time.
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Figure 3.
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LTP, low ellagitannin level; MTP, medium ellagitannin level; HTP, high ellagitannin level. 160, toasted at 160 °C as initial temperature; 180, toasted at 180 °C as initial temperature. Different letters in the bars correspond to significant differences between the oak barrel modalities.
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Figure 4.
LTP, low ellagitannin level; MTP, medium ellagitannin level; HTP, high ellagitannin level. 160, toasted at 160 °C as initial temperature; 180, toasted at 180 °C as initial temperature. Different letters in the bars correspond to significant differences between oak barrel modalities for each winery at a given time.
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Highlights Malvidin coumaroyl glucoside is the most reactive anthocyanin during barrel aging
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Low ellagitannin barrels accelerate the formation of stable wine pigments
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Oak barrel modalities did not impact on the wine color intensity at one year
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