Effect of wine micro-oxygenation treatment and storage period on colour-related phenolics, volatile composition and sensory characteristics

Effect of wine micro-oxygenation treatment and storage period on colour-related phenolics, volatile composition and sensory characteristics M.J. Cejudo-Bastante a , * , M.S. Pérez-Coello a , I. Hermosín-Gutiérrez b a Área de Tecnología de los Alimentos, Facultad de Ciencias Químicas, Avda. Camilo José Cela s/n, 13071 Ciudad Real, Spain b IRICA (Instituto Regional de Investigación Científica Aplicada), Escuela Universitaria de Ingeniería Técnica Agrícola, Universidad de Castilla-La Mancha, Ronda de Calatrava 7, 13071 Ciudad Real, Spain article info Article history: Received 9 June 2010 Received in revised form 13 October 2010 Accepted 14 October 2010 Keywords: Micro-oxygenation Volatile compounds Polyphenols Pigments Sensorial analysis Storage abstract In this paper, we have evaluated the effects of micro-oxygenation before malolactic fermentation and after five months of storage on Cencibel red wines. In particular, we have considered the colour characteristics, the phenolic compounds related to red wine colour, the individual volatile composition, and the complete descriptive sensory analysis of the wines. The fact that the concentration of the malvidin-3glucoside-ethyl-flavan-3-ol adducts and pyranoanthocyanins (B-type vitisins) increased is closely related to the red wine colour stabilization. Red wine aroma quality was slightly improved as a consequence of oxygen addition after five months of storage. New attributes appeared (plum/currant) and others were increased (spicy and liquorice) in micro-oxygenated red wines, whereas herbaceous values were significantly decreased. The results suggest the joint use of both treatments (micro-oxygenation and storage) give rise to an enhancement of the colour stability and the aroma and sensorial quality of red wines. Ó2010 Elsevier Ltd. All rights reserved. 1. Introduction The micro-oxygenation treatment produces a stabilization of red wine colour and improves the wine quality by the addition of small, continuous and controlled quantities of oxygen. The microoxygenation concept was born by the investigation of Patrick Ducournau and Laplace family in 1993, and is based on the colour stabilization and astringency diminution that normally occurred in oak barrels. The application of oxygen before malolactic fermentation has been described by several authors as the optimal moment (Hernández-Orte et al., 2009; Ortega-Heras, Rivero-Pérez, Pérez-Magariño, González-Huerta, & González-Sanjosé, 2008). On one hand, the oxygen is involved in several wine reactions, mainly those involving phenolic compounds. As a consequence, new products result from the direct and acetaldehyde-mediated anthocyaninetannin reactions (Escribano-Bailón, Álvarez-García, RivasGonzalo, Heredia, & Santos-Buelga, 2001). Pyranoanthocyanin formation by condensation of anthocyanins with other molecules having polarizable double bond is also possible (Atanasova, Fulcrand, Cheynier, & Moutounet, 2002; Rentzsch, Schwarz, Winterhalter, & Hermosín-Gutiérrez, 2007). These compounds are more stable than genuine anthocyanins, leading to red wine colour stabilization by condensation reactions. In spite of the scarce studies about micro-oxygenation effects on the volatile compounds, Ortega-Heras et al. (2008) assert that the results depend to a large extent on the grape cultivar and vintage. The differences between micro-oxygenated and untreated wines in terpenes, esters and acetates concentration disappeared after malolactic fermentation (Hernández-Orte et al., 2009; OrtegaHeras et al., 2008), contrarily to that found by Cerdán, Goni, and Azpilicueta (2004) in esters composition. Moreover, the theoretical expected diminution of green and herbaceous aromas in microoxygenated red wines, described by several authors (Bertuccioli, Rosi, Lencioni, Zini, & Siliani, 2001), does not seem to be correlated with the six-carbon alcohols concentration decrease (OrtegaHeras et al., 2008). On the other hand, red wine ageing in bottle produces different effects, like colour stability improvement, spontaneous clarification and more complex and stabilized phenolic pigments (Cruz et al., 2008). Polymerization and condensation reactions occurred during ageing, mainly between anthocyanins and flavan-3-ols (anthocyanin-flavan-3-ol adducts, by direct reaction or acetaldehyde-mediated reaction), and also other phenolic and non-phenolic compounds (formation of pyranoanthocyanins and hydroxyphenylpyranoanthocyanins) (Francia-Aricha, Guerra, Rivas-Gonzalo, & Santos-Buelga, 1997). As a consequence, the red-purple colour *Corresponding author. Tel.: þ34 926295300 3425; fax: þ34 926295318. E-mail address: maria[email protected] (M.J. Cejudo-Bastante). Contents lists available at ScienceDirect LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt 0023-6438/$ esee front matter Ó2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2010.10.015 LWT - Food Science and Technology 44 (2011) 866e874 gradually disappears, giving rise to reddish-brown hue (Atanasova et al., 2002). Also, ageing red wines improves the aroma and taste quality due to the diminution of astringency (Puech, Feuillat, Mosedale, & Puech, 1996). Although the aroma stabilization during storage greatly depends on the grape variety, the floral and fruity aromas produced by monoterpenes and acetates and esters, respectively, decrease after bottling (Pérez-Coello, Martín-Álvarez, & Cabezudo, 1999). The effect on wine colour-related phenolic compounds,volatilecomposition andsensorycharacteristicsduring storage is well-known, but the combined effect of wine microoxygenation and storage has not been previously reported. The aim of this research was to study the effects of microoxygenation and later storage on the colour parameters, phenolic and volatile composition and subsequently descriptive sensorial characteristics. The study has been performed in Cencibel red wines, a large extent cultivar in Castilla-La Mancha (Spain), developing a complete study not previously reported. 2. Material and methods 2.1. Winemaking Red wine made from Vitis vinifera grape cv. Cencibel (harvested at the optimal maturity stage and in good sanitary conditions) was elaborated in the experimental winery of Castilla-La Mancha University (in central-southeast Spain), following widespread winemaking methodology. After manual harvest, the mass obtained was sulphited (80 mg/L of total SO 2 ; 29 mg/L of free SO 2 ), destemmed and crushed. Fermentations were carried out in duplicate after inoculation with Saccharomyces cerevisiae race cerevisiae yeasts (CECT No. 10835) with skin maceration. Fermentations were conducted at controlled temperature (24e26  C). After alcoholic fermentation, and previous to malolactic fermentation, the wine was micro-oxygenated. Inhibition of the development of malolactic fermentation during oxygen treatment was achieved by addition of 20 g/HL of lysozyme. The wine was homogeneously distributed within 4 stainless steel tanks of 2000 L of capacity and 2 m of height, which guarantee the complete oxygen dissolution in wine during the micro-oxygenation treatment. Two tanks were submitted to micro-oxygenation treatment and the other two tanks contained untreated, non-micro-oxygenated control wine. The micro-oxygenation treatment consisted in an oxygen dose of 10 mL/L/month during 20 days at 20  C, by means of a microdiffusion system (Laffort, Spain). The dose supplied were appropriated according to the wine total polyphenol index (TPI ¼65-70) and the manufacturer recommendations. After micro-oxygenation treatment, the red wines were inoculated with 1g/HL of a commercial lactic acid bacteria strain of Oenococcus Oeni (Lactobacter SP1; Laffort, Spain). The development of malolactic fermentation was monitored by TLC and malic acid and lactic acid enzymatic measurement. The wine characteristics were monitored at different moments of the process: after microoxygenation treatment, after malolactic fermentation completion (only for the colour-related phenolics) and after subsequent five months of storage in stainless steel vats (16  C of temperature and dark conditions). All the sample replicates were analyzed in duplicate. Wine conventional analytical data and development of malolactic fermentation were obtained by O.I.V. official methods (1990). 2.2. Analysis of wine polyphenolic compounds and colour parameters A Hewlett Packard 8452A apparatus were used for the analysis of main phenolic types by spectrophotometry. Total polyphenolic, anthocyanins, hydroxycinnamic acid derivatives and flavonols families (Mazza, Fukumoto, Delaquis, Girard,& Ewert,1999), flavan3-ols family (Amerine & Ough, 1980), and tannins (Glories, 1988) have been measured. Also, the chromatic characteristics in the CIELAB space (Pérez Caballero, Ayala, Echávarri, & Negueruela, 2003)L * , C*, h*, a*, b* and colour parameters (colour intensity and tonality) (Glories, 1984) were calculated. The method described by Hermosín-Gutiérrez (2003) was used for the determination of the percentage contributions of copigmented and polymerized anthocyanins to the total wine colour at pH 3.6. HPLC separation, identification and quantification of phenolic compounds were performed on an Agilent 1100 series system (Agilent, Waldbronn, Germany), equipped with a DAD photodiode detector (G1315B) and an LC/MSD Trap VL (G2445C VL) electrospray ionization mass spectrometry (ESI/MS n ) system, according to the method proposed by Castillo-Muñoz, Gómez-Alonso, GarcíaRomero, and Hermosín-Gutiérrez (2007). On one hand, direct injection of the must and wine samples were used for the analysis of the anthocyanins, benzoic acid derivatives and flavan-3-ols compounds, and quantification was made using the DAD-chromatograms obtained at 520 and 280 nm, respectively. Monomeric anthocyanins and anthocyanin-ethyl-flavan-3-ols were quantified using the calibration curve of malvidin-3-glucoside, whereas the calibration curve of pinotin A was used to quantify pyranoanthocyanins and hydroxyphenyl-pyranoanthocyanins. In the case of benzoic acids and flavan-3-ols, individual calibration curves were employed to quantify each individual compound. On the other hand, anthocyanins-free extracts were obtained by SPE on Oasis MCX cartridges for the isolation of red wine flavonols and hydroxycinnamic acid derivatives, according to Castillo-Muñoz et al. (2007). The chromatographic method used was that proposed by Castillo-Muñoz et al. (2009). Quantification was made using the DAD-chromatograms obtained 360 and 320 nm, respectively. Individual calibration curves were obtained for each flavonol, with some exceptions: on the one hand, myricetin and laricitrin 3-glycosides were quantified as myricetin-3-glucoside and, on the other hand, laricitrin, kaempferol 3-glycosides and quercetin-3galactoside were quantified as myricetin, kaempferol-3-glucoside and quercetin-3-glucoside, respectively. Similarly, individual calibration curves were used to quantify each hydroxycinnamic acid and their respective esters. For identification, the ESI-MS n was used in positive mode for anthocyanins and flavan-3-ols, whereas both positive and negative modes were used for flavonols and hydroxycinnamic acid derivatives. 2.3. Analysis of wine volatile compounds After centrifugation, samples were directly injected in a Hewlett Packard 5890 Series II Gas Chromatograph, coupled to a flame ionization detector, according to the method proposed by SánchezPalomo, González-Viñas, Díaz-Maroto, Soriano-Pérez, and PérezCoello (2007) for the determination of major volatile compounds. Minor volatile compounds of wines were extracted in duplicate by Solid Phase Extraction (SPE) technique, according to the method proposed by Sánchez-Palomo, Alañón, Díaz-Maroto, GonzálezViñas, and Pérez-Coello (2009). The extract was injected into an Agilent Technology 6890N Network GC System equipped with an Agilent Technology 5973 inert Mass Selective Detector, according to the method proposed by Sánchez-Palomo et al. (2007). NBS75K and Wiley A libraries were used for compounds identification, together with the comparison of the GC retention times and mass spectra of the pure substances. The response factor of each compound was experimentally obtained by injection of commercial standards. For compounds without reference compounds available, the response factors of standards with similar chemical structures were used. M.J. Cejudo-Bastante et al. / LWT - Food Science and Technology 44 (2011) 866e874 867 2.4. Descriptive sensory analysis The descriptive sensorial analysis was carried out by a group of expert assessors with previous experience in sensoryanalysis. After training in wine descriptive sensory analysis, Cencibel red wines were tested. Assessment took place in a standard sensory-analysis chamber (ISO 8589-1998), equipped with separate booths, and a wine-testing glasses (ISO 3591-1997) covered with a watch-glass to minimize the escape of volatile compounds. Wines were sniffed and tasted. Panellists used a 10 cm unstructured scale to rate the intensity of each attribute. The wine samples were evaluated in duplicate. 2.5. Statistics The SPSS version 15.0 for Windows statistical package was used for statistical processes. The Student’sttest was applied to discriminate among the means of chemical data by pairs of nonmicro-oxygenated control and micro-oxygenated wines for each moment. Furthermore, a Principal Component Analysis (PCA) was carried out with the aim of highlighting the main contributors to the variance among wines in different moments of the process. 3. Results and discussion 3.1. General parameters The low values for reducing sugars, fructose and glucose indicated the correct development of alcoholic fermentation, being considered as dry red wines (reducing sugars below 5 g/L) (Table 1). Also, the malolactic fermentation development was optimal, due to Table 1 Conventional analysis of Cencibel red wines (NMC, non-micro-oxygenated control wine; M, micro-oxygenated wine): after micro-oxygenation treatment (MOX) and subsequently malolactic fermentation (MLF). After MOX After MLF NMC M NMC M total acidity (g/L) 6.44 6.34 4.83 4.75 volatile acidity (g/L) 0.41 0.33 0.6 0.53 pH 3.79 3.79 3.96 3.97 density (g/L) 994 994 993 993 free SO 2 (mg/L) 26.0 30.1 37.4 24.3 total SO 2 (mg/L) 78.2 81.3 96.1 98.4 alcoholic strengh (%vol) 14.5 14.5 14.6 14.6 reducing sugars (g/L) 1.79 1.92 1.64 <1.50 fructose (g/L) <1.20 <1.20 <1.20 <1.20 glycerin (g/L) 8.41 8.32 9.48 9.32 glucose (g/L) <0.20 <0.20 <0.20 <0.20 acetic acid (g/L) 0.25 0.15 0.44 0.39 gluconic acid (g/L) nd nd nd nd tartaric acid (g/L) 2.92 3.06 2.95 3.04 L -lactic acid (g/L) 0.74 0.75 1.94 1.96 malic acid (g/L) 1.95 1.92 <0.10 <0.10 TPI a 65.3 66.1 70.3 71.2 nd, non detected. a TPI, total polyphenolic index. Table 2 Mean values of concentration (mg/L) and standard deviations (n¼2) of non-anthocyanic phenolic compounds belonged to different chemical families (hydroxycinnamic acid derivatives (HCAD), benzoic acids derivatives, flavan-3-ols and flavonols) identified by HPLC-MS in non-micro-oxygenated control (NMC) and micro-oxygenated (M) Cencibel red wines: after micro-oxygenation treatment (MOX), after malolactic fermentation (MLF) and subsequently 5 months of storage. After MOX a After MLF 5 Months of Storage NMC M NMC M NMC M HCAD tecaftaric acid 65.3 1.01 65.4 0.37 31.9 0.04 27.2 9.70 10.1 0.13 8.81 0.00* tecoutaric acid 32.2 0.95 32.9 0.18 15.2 0.05 12.4 4.99 3.84 0.12 3.10 0.00 cecoutaric acid 9.49 0.01 9.38 0.06 4.53 0.05 3.78 1.62 1.11 0.05 0.92 0.01 tefertaric acid 6.33 0.15 6.33 0.06 2.93 0.06 2.17 0.88 1.45 0.06 1.18 0.01 cefertaric acid 8.83 0.18 8.84 0.04 2.11 0.14 1.50 0.63 1.40 0.42 1.40 0.00 caffeic acid nd nd 20.5 0.09 15.2 5.27 29.2 1.11 29.9 0.15 p-coumaric acid 1.85 0.02 1.93 0.02 9.36 0.07 6.58 1.95 13.7 0.37 15.3 0.05 ferulic acid 0.39 0.01 0.35 0.00 0.89 0.02 0.73 0.02* 1.44 0.20 1.36 0.00 Benzoic acids gallic acid 7.57 0.33 8.26 0.02 9.04 2.25 6.01 0.33 10.9 0.30 10.9 1.19 Flavan3ols (+)-catechin 16.3 0.61 16.0 0.24 15.3 0.55 13.5 0.55 16.1 0.18 14.8 0.48 ()-epicatechin 9.18 0.48 8.92 0.05 7.80 0.15 7.31 0.25 7.78 0.01 8.75 1.73 ()-epicatechin gallate ester nq nq nq nq nq nq Flavonols myricetin3galactoside 1.72 0.18 1.86 0.02 1.46 0.08 1.38 0.06 1.37 0.08 1.37 0.04 myricetin3glucuronide 1.77 0.05 1.79 0.16 1.59 0.06 1.29 0.05 1.37 0.03 1.21 0.10* myricetin3glucoside 48.2 0.37 49.7 0.20 42.3 1.81 42.8 3.51 39.3 .83 39.2 0.56 quercetin3galactoside 1.55 0.02 1.66 0.03* 1.35 0.05 1.40 0.07 1.29 0.16 1.18 0.01 quercetin3glucuronide 6.50 0.37 6.54 0.08 5.48 0.41 5.03 0.58 4.75 0.02 4.74 0.24 quercetin3glucoside 18.1 0.04 18.1 0.09 15.5 0.45 15.5 0.91 14.0 0.39 14.5 0.69 kaempferol3galactoside 0.57 0.07 0.62 0.01 0.53 0.01 0.42 0.04 0.25 0.02 0.40 0.01* kaempferol3glucuronide 0.16 0.02 0.45 0.01* 0.32 0.01 0.20 0.01* 0.22 0.02 0.25 0.01 kaempferol3glucoside 3.92 0.13 4.10 0.06 3.33 0.11 3.34 0.20 3.29 0.26 2.86 0.04 laricitrin3eglucoside 9.82 0.25 10.0 0.04 8.65 0.17 8.64 0.52 7.89 0.31 7.86 0.07 isorhamnetin3eglucoside 2.20 0.05 2.21 0.02 1.95 0.01 1.89 0.13 1.77 0.08 1.78 0.01 syringetine3-glucoside 6.06 0.35 6.30 0.00 5.62 0.09 5.49 0.41 5.10 0.19 5.05 0.06 quercetin 0.76 0.09 0.96 0.18 0.83 0.13 0.62 0.02 1.04 0.01 0.42 0.08* kaempferol 0.19 0.00 0.19 0.00 0.23 0.02 0.16 0.02 0.21 0.01 0.15 0.02 laricitrin 0.15 0.01 0.16 0.00 0.16 0.02 0.10 0.01 0.15 0.01 0.08 0.00* isorhamnetin 0.18 0.01 0.19 0.01 0.15 0.02 0.11 0.01 0.23 0.03 0.23 0.03 syringetin 0.31 0.00 0.33 0.01 0.22 0.02 0.21 0.02 0.29 0.04 0.35 0.03 nq, not quantificable. a Asterisks denote significant differences according to the Student’s test of p<0.05 between non-micro-oxygenated control (NMC) and micro-oxygenated (M) wines. t, trans; c, cis; HCAD, hydroxycinnamic acid derivatives; GRP, Grape Reaction Product (2-S-glutathionyl caftaric acid). M.J. Cejudo-Bastante et al. / LWT - Food Science and Technology 44 (2011) 866e874868 the almost complete transformation of malic acid to lactic acid. The expected loss of total acidity was observed together with the also expected increase of volatile acidity, although below the CEE limit (1.2 g/L). The micro-oxygenation treatment did not showsignificant effects on the conventional analysis. 3.2. Phenolic compounds identified in Cencibel red wines Many types of phenolic compounds were identified in nonmicro-oxygenated control and micro-oxygenated red wines: benzoic acids and hydroxycinnamic acid derivatives, flavan-3-ols, flavonols, anthocyanins and anthocyanin-related red pigments (Tables 2 and 3). Grape native anthocyanins, flavonol 3-glycosides and free flavonol aglycones found in our Cencibel red wines were in agreement with previously reported data (Alcalde-Eón, Boido, Carrau, Dellacassa, & Rivas-Gonzalo, 2006b; Castillo-Muñoz et al., 2007; Hermosín Gutiérrez & García-Romero, 2004). Among the pyranoanthocyanins, the following compounds were identified by ESI-MS n and quantified by DAD: the complete series of non-acylated,acetylatedandp-coumaroylatedB-typevitisinsandthe non-acylatedandp-coumaroylatedA-typevitisins,bothderivedfrom malvidin; and the B-type vitisin derived from peonidin. Other pyranoanthocyanins (acetylated B-type vitisin from petunidin and pcoumaroylated B-type vitisin from peonidin) were identified on the basisoftheirESI-MS n data(Fig.1a1,1a2,1b1 and1b2)buttheydidnot give quantificable peaks in the DAD-chromatograms. Withregards to hydroxyphenyl-pyranoanthocyanins, malvidin-3-glucoside-4-vinylcatechol(alsoknownaspinotinA)andmalvidin-3-glucoside-4-vinylphenol were detected by ESI-MS n in Cencibel red wines, although pinotinAwasnotquantificablebecauseofthelowintensepeakitgave in the DAD-chromatograms, even after the storage period. The low concentration of pinotin A was in agreement with recently reported data (Rentzsch, Schwarz, Winterhalter, Blanco-Vega, & HermosínGutiérrez, 2010) for young Tempranillo wines. It is worth mentioning the detection and quantification of several minor red pigments in Cencibel wines derived from the reaction of malvidin-3-glucoside and flavan-3-ols, mediated by acetaldehyde (ethyl bridged). Firstly, the four possible isomers of malvidin-3glucoside-ethyl-flavan-3-ol were identified and quantified (Table 3), showing a maximum absorbance at 539 nm with an additional shoulderat 455 nm and giving a molecularionsignal at m/z 809 (data not shown). The latter compounds had been previously reported in some red wines from Portugal, Spain and Uruguay (Alcalde-Eón, Escribano-Bailón, Santos-Buelga, & Rivas-Gonzalo, 2004, 2006a; Table 3 Mean values of concentration (mg/L) and standard deviations (n¼2) of anthocyanic phenolic compounds belonged to different chemical families (monomeric anthocyanins, anthocyanin-flavan-3-ol addutcs mediated by acetaldehyde, pyranoanthocyanins and hydroxyphenyl-pyranoanthocyanins) identified by HPLC-MS in non-micro-oxygenated control (NMC) and micro-oxygenated (M) Cencibel red wines: after micro-oxygenation treatment (MOX), after malolactic fermentation (MLF) and subsequently 5 months of storage. After MOX a After MLF 5 Months of Storage NMC M NMC M NMC M Monomeric anthocyanins delphinidin-3-glucoside 14.4 0.09 15.0 0.01 12.6 0.28 11.1 0.34* 11.5 0.04 10.7 0.23 cyanidin3glucoside 7.45 0.02 7.63 0.01* 7.27 0.05 6.97 0.06* 7.01 0.01 6.83 0.03* petunidin3glucoside 22.7 0.16 24.6 0.08* 20.6 0.56 16.9 0.80* 17.2 0.06 15.1 0.30* peonidin3glucoside 14.0 0.28 14.8 0.01 12.4 0.15 10.6 0.24* 11.3 0.07 10.0 0.09* malvidin3glucoside 126 1.72 139 0.43* 108 3.28 79.4 3.91* 90.1 0.31 70.7 1.50* delphinidin3acetyleglucoside 8.41 0.27 8.33 0.03 8.10 0.06 7.77 0.03* 8.00 0.04 7.68 0.03* petunidin3acetyleglucoside 9.43 0.01 9.65 0.06 9.17 0.11 8.61 0.03 8.63 0.08 8.44 0.05 peonidin3acetyleglucoside 7.85 0.06 8.03 0.13 7.77 0.02 7.46 0.01* 7.50 0.01 7.34 0.00* malvidin3acetyleglucoside 19.9 0.06 21.1 0.03 18.9 0.32 14.9 0.27* 16.7 0.01 14.4 0.07* cedelphinidin3p-coumaroyleglucoside 8.69 0.09 9.14 0.15* 8.60 0.01 8.65 0.01 8.72 0.03 8.75 0.01 tedelphinidin3p-coumaroyleglucoside 12.6 0.49 13.1 0.05 13.0 0.13 10.7 0.07* 12.1 0.03 10.8 0.03* t-cyanidin3pecoumaroyleglucoside 7.32 0.03 7.32 0.03 7.33 0.06 6.84 0.04* 7.06 0.01 6.82 0.01* c-petunidin3p-coumaroyleglucoside 9.54 0.21 10.3 0.45 9.28 0.01 8.93 0.02* 9.56 0.03 9.47 0.00 t-petunidin3p-coumaroyleglucoside 13.2 0.00 13.4 0.39 13.0 0.12 10.8 0.13 12.2 0.04 10.9 0.05* c-peonidin3p-coumaroyleglucoside 8.60 0.07 9.13 0.11* 8.51 0.01 8.48 0.06* 8.70 0.02 8.59 0.01 t-peonidin3p-coumaroyleglucoside 12.6 0.11 12.5 0.25 11.9 0.09 10.4 0.11* 11.4 0.02 10.3 0.03* c-malvidin3p-coumaroyleglucoside 12.2 1.37 16.8 1.73 9.60 0.82 9.39 0.50 12.7 0.19 11.7 0.07* t-malvidin3p-coumaroyleglucoside 47.6 1.33 48.4 1.95 43.7 0.99 27.5 0.95* 37.7 0.01 27.9 0.26* malvidin3caffeoyleglucoside 9.97 0.04 9.98 0.05 10.0 0.08 9.47 0.04* 9.70 0.00 9.47 0.01* Pyranoanthocyanins A-type vitisin (malvidin) 4.06 0.03 3.70 0.04* 2.54 0.09 1.71 0.02* 2.12 0.02 1.85 0.00* p-coumaroyl A-type vitisin (malvidin) 2.62 0.53 3.95 0.13 1.98 0.04 1.08 0.05* 2.67 0.08 2.46 0.01 B-type vitisin (peonidin) 0.77 0.04 0,75 0.03 0.57 0.01 0.74 0.02* 0.39 0.00 0.35 0.01 B-type vitisin (malvidin) 3.97 0.06 2.66 0.12* 2.62 0.10 4.69 0.02* 1.18 0.01 2.15 0.02* acetyl B-type vitisin (malvidin) 0.62 0.04 0.49 0.00 0.52 0.02 1.02 0.00* 0.69 0.09 0.63 0.00 p-coumaroyl B-type vitisin (malvidin) 1.86 0.10 1.56 0.12 1.90 0.17 2.06 0.19 0.84 0.03 1.35 0.06* Hydroxyphenylepyranoanthocyanins malvidin3glucoside4vinylphenol 0.45 0.01 0.49 0.00 0.63 0.03 0.33 0.02* 0.69 0.00 0.61 0.00* Anthocyanineethyleflavan3ol petunidin3glucosideeethyleflavan3ol 11.1 0.07 11.0 0.01 11.0 0.00 11.2 0.06 11.0 0.05 11.1 0.03 malvidin3glucosideethyleflavan3ol 1 11.4 0.11 11.2 0.01 11.3 0.00 11.6 0.01* 11.2 0.01 11.3 0.00* malvidin3glucosideethyleflavan3ol 2 12.5 0.01 12.1 0.03* 12.2 0.01 12.8 0.06* 12.0 0.00 12.0 0.03 malvidin3glucosideethyleflavan3ol 3 12.4 0.04 12.2 0.08 12.4 0.02 13.3 0.14 12.0 0.07 12.6 0.05* malvidin3glucosideethyleflavan3ol 4 12.2 0.01 12.1 0.05 12.2 0.01 12.6 0.08 11.6 0.01 12.1 0.02* malvidin3p-coumaroyleglucosideeethyleflavan3ol 1 13.8 0.07 13.8 0.02 13.5 0.01 13.5 0.00 14.0 0.00 14.0 0.02 malvidin3p-coumaroyleglucosideeethyleflavan3ol 2 13.4 0.01 13.4 0.00 13.2 0.00 13.3 0.00* 13.4 0.00 13.7 0.02* malvidin3p-coumaroyleglucosideeethyleflavan3ol 3 13.3 0.01 13.3 0.02 13.3 0.00 13.2 0.02 13.2 0.00 13.2 0.00 t, trans; c, cis. a Asterisks denote significant differences according to Student’s test (p<0.05) between non-micro-oxygenated control and micro-oxygenated red wines. M.J. Cejudo-Bastante et al. / LWT - Food Science and Technology 44 (2011) 866e874 869 Alcalde-Eón, Boido, Carrau, Dellacassa, & Rivas-Gonzalo, 2006b; Boido, Alcalde-Eón, Carrau, Dellacassa, & Rivas-Gonzalo, 2006). Secondly, three of the four possible isomers of malvidin-3-p-coumaroyl-glucoside-ethyl-flavan-3-ol were also identified (visible maximum at 550 nm and molecular ion signal at m/z 955, data not shown)andquantified(Table 3)inCencibelredwines.Onlytwoofthe aforementioned isomers had been previously reported (Alcalde-Eón et al., 2004; Boido et al., 2006). In addition, the four possible isomers of malvidin-3-acetyl-glucoside-ethyl-flavan-3-ol were also identified (molecular ions at m/z851 and MS 2 fragmentation pattern asexpected,Fig.1c1and1c2),buttheywere notquantifiablebyDADchromatograms; previously reported data only referred to two of these isomers (Boido et al., 2006). Finally, two anthocyanin-ethylflavan-3-ol adducts were identified by ESI-MS n , the data suggesting they were petunidin-3-glucoside-ethyl-flavan-3-ol (molecular ion at m/z 795andMS 2 fragmentationpatternasexpected;datanotshown) and peonidin-3-glucoside-ethyl-flavan-3-ol (molecular ion at m/z 779, Fig. 1d1and 1d2). Only petunidin-3-glucoside-ethyl-flavan-3-ol was quantifiable by DAD-chromatograms. 3.3. Micro-oxygenation effect on phenolic composition and colour parameters After oxygen addition, micro-oxygenated and non-microoxygenated control red wines had significant differences in chromatic characteristics, according to the Student’sttest, which almost disappeared after malolactic fermentation, when micro-oxygenated wines only maintained the lowest chroma values (C*) (Table 4). According to that reported for aged Tannat red wines from Uruguay (Boido et al., 2006), a decrease in the contribution of the red colour component (a*) and an increase of the yellow colour component (b*) was observed after five months of storage. At this moment, stored micro-oxygenated red wines had significant higher values of chroma (C*) and higher contribution of the red and yellow colour components (a* and b*, respectively) at pH 3.6, in comparison with non-micro-oxygenated control wines. Only punctual significant differences were observed between non-micro-oxygenated control and micro-oxygenated red wines in each moment for the content of hydroxycinnamic acid derivatives +MS2(545.6), 21.9min #721 0 2 4 6 5 x10 200 400 600 800 1000 m/z 325.1 355.1 +MS2(633.6), 27.2min #905 0 2 4 5 x10 200 400 600 800 1000 m/z 357.1 561.1 647.2 +MS2(851.7), 26.8min #889 0 2 4 6 4 x10 200 400 600 800 1000 m/z 327.1 489.1 617.1 +MS2(779.7), 21.2min #695 0 1 2 3 4 x10 200 400 600 800 1000 m/z MICROOX000137.D: EIC 545 +All MICROOX000137.D: EIC 633 +All MICROOX000137.D: EIC 851 +All MICROOX000137.D: EIC 779 +All 0 2 5 x10 Intens. 0 1 2 5 x10 0.0 0.5 5 x10 0 2 4 4 x10 5 10 15 20 25 30 35 40 Time [min] 18.8 27.2 26.3 26.8 28.4 30.8 21.2 Fig. 1. HPLC chromatograms corresponding to the fraction of non-quantificable compounds by UVevis and identified by MS: EIC chromatograms (ESI-MS and MS 2 modes) for detection of B-type vitisins from petunidin-3-acetyl-glucoside (a1, EIC 545 þAll and a2, þMS 2 545) and peonidin-3-p-coumaroyl-glucoside (b1, EIC 633 þAll and b2, þMS 2 633) and for the adducts malvidin-3-acetyl-glucoside-ethyl-flavan-3-ol (c1, EIC 851 þAll and c2, þMS 2 851) and peonidin-3-glucoside-ethyl-flavan-3-ol (d1, EIC 779 þAll and d2, þMS 2 779). M.J. Cejudo-Bastante et al. / LWT - Food Science and Technology 44 (2011) 866e874870 and flavan-3-ols, as individual components or global concentrations (Tables 2 and 4). Similar results were observed by Castellari, Matricardi, Arfelli, Galassi, and Amati (2000) and Pérez-Magariño, Sánchez-Iglesias, Ortega-Heras, González-Huerta, and GonzálezSanjosé (2007) in Mencía and Tempranillo red wines. In the case of total flavan-3-ols, micro-oxygenated red wines showed significant lower concentration after malolactic fermentation, which was in agreement with the significant higher percentage of contribution to red wine colour of polymerized anthocyanins (% polymerization) in this wine (Table 2)(Castellari et al., 2000). Anthocyanins and anthocyanin-related red pigments were the main phenolic compounds that were significantly affected by micro-oxygenation treatment, according to ANOVA Student’sttest (Table 3). The individual monomeric anthocyanins concentration just after microoxygenation treatment was not so much affected by the addition of oxygen, in contrast to the decreasing effect observed after malolactic fermentation and also after subsequent period of storage (Table 3), in agreement with reported results (Cano-López, Pardo, López-Roca, & Gómez-Plaza, 2006; Pérez-Magariño et al., 2007). The significant diminution in monomeric anthocyanin concentration due to the micro-oxygenation treatment, together with the aforementioned lower content of flavan-3-ols, suggest that the oxygen addition activated the reactions between free anthocyanins and flavan-3-ols. As a consequence, new anthocyanin-derived pigments more stable to pH changes and bisulphite bleaching were formed (Escribano-Bailón et al., 2001). The latter suggestion was supported by the already mentioned significant increase of % polymerization in micro-oxygenated red wines after malolactic fermentation, and also by the lower value of the contribution to red wine colour of copigmentated anthocyanins (% copigmentation) and the colour intensity, which can be easily explained by the lower concentration of monomeric anthocyanins (Hermosín-Gutiérrez, Sánchez-Palomo & Vicario-Espinosa, 2005). The observed decrease of colour intensity is negatively correlated with lightness (L*), according to several authors (Hermosín, 2003). With regard to anthocyanin-related pigments, the concentrations of B-type vitisins and anthocyanin-ethyl-flavan-3-ols adducts significantly increased after malolactic fermentation as a consequence of oxygen addition, in contrast to A-type vitisins. However, control wines did not show significant changes in red wine pigments, and only a slight decrease in anthocyanin-derived pigments was observed. The above mentioned results support the hypothesis that added oxygen induced in micro-oxygenated wines the oxidation of ethanol giving rise to acetaldehyde, that further directly reacted with anthocyanins (B-type vitisins formation) or mediated the reaction between anthocyanins and flavan-3-ols (formation of anthocyanin-ethyl-flavan-3-ol adducts). After five months of storage, the concentrations of B-type vitisins, hydroxyphenyl-pyranoanthocyanins and anthocyanin-ethylflavan-3-ol adducts decreased for both non-micro-oxygenated control and micro-oxygenated wines. With regards to anthocyaninethyl-flavan-3-ols adducts, it is known they are labile compounds that break giving rise to anthocyanins and 8-vinyl-flavan-3-ols (Escribano-Bailón et al., 2001). In addition, non-micro-oxygenated control wines also experienced a decrease of the content of monomeric anthocyanins. Although the loss of anthocyanin-related pigments (B-type vitisins, hydroxyphenyl-pyranoanthocyanins and anthocyanin-ethyl-flavan-3-ol adducts) was higher for microoxygenated wines in comparison to non-micro-oxygenated control wines, the final concentration of such red pigments was still significantly higher in micro-oxygenated wines after the storage period, according to ANOVA Student’sttest (Table 3). According to Sartini,Arfelli,Fabiani,andPiva(2007),theformation ofB-typevitisinsandhydroxyphenyl-pyranoanthocyaninsareclosely related with the increasing of the yellow colour component (b*)to aged red wines and they are considered more stable pigments than monomeric anthocyanins, thus contributing to red wine colour stabilization. Therefore, it can be suggested that micro-oxygenation treatment produced colour stabilization in Cencibel red wines that is maintained during a storage period of five months. 3.4. Micro-oxygenation effect on volatile compounds A total of 94 individual volatile compounds have been identified in Cencibel red wines (data not shown) structured in different families: esters, lactones, terpenes, aldehydes, alcohols, acids, benzenic compounds and pyrazines (Table 5). As a consequence of storage, an increase of esters and lactones concentration was observed for both wines as well as a slight increase in benzenic compounds. According to several authors (González-Viñas, Pérez-Coello, Salvador, Cabezudo, & MartínÁlvarez, 1996; Pérez-Coello et al., 1999), succinic acid derivatives, large-chain esters and several hydroxy-esters were increased after storage conditions, due to the reductive conditions. Contrarily, Table 4 Chromatic characteristics, global types of polyphenol families (g/L), and colour parameters by spectrophotometic measures of Cencibel red wines (mean value standard deviation; n¼2): after micro-oxygenation treatment (MOX), after malolactic fermentation (MLF) and subsequently five months of storage. After MOX a After MLF 5 Months of Storage NMC M NMC M NMC M L* 44.3 0.00 45.00 0.00 51.9 0.00 54.4 0.00 55.7 0.00 53.3 0.07* C* 53.9 0.04 54.90 0.04* 47.1 0.01 43.8 0.01* 44.1 0.06 44.4 0.05* h* 4.76 0.10 3.20 0.04* 5.19 0.03 5.39 0.08 6.15 0.06 6.44 0.01 a* 53.7 0.04 54.81 0.04* 46.9 0.01 45.1 2.11 43.8 0.06 44.1 0.05* b* 4.47 0.08 3.07 0.04* 4.26 0.02 4.12 0.06 4.72 0.04 4.97 0.01* colour intensity 12.9 0.00 12.61 0.02* 10.0 0.00 9.16 0.00* 8.89 0.01 9.49 0.01* tonality 2.82 0.00 2.72 0.00* 3.10 0.00 3.14 0.00* 3.19 0.00 3.15 0.00* copigmentation (%) 33.9 1.7 40.8 1.3* 21.6 1.1 20.1 1.0 32.3 0.8 25.6 1.1* polymerization (%) 44.9 1.8 43.2 1.1 46.1 0.7 54.0 0.4* 51.1 0.2 56.4 1.1 tannins 1.55 0.08 1.54 0.03 1.69 0.05 1.48 0.22 1.18 0.04 1.12 0.04 total polyhenols 1.54 0.00 1.58 0.04 1.33 0.047 1.16 0.02 1.16 0.01 0.99 0.09 HCAD 0.42 0.03 0.43 0.01 0.39 0.01 0.36 0.05 0.35 0.01 0.31 0.02 flavonols 0.23 0.00 0.24 0.01 0.20 0.00 0.18 0.00 0.18 0.00 0.16 0.01 anthocyanins 0.54 0.01 0.58 0.02 0.44 0.01 0.38 0.01* 0.39 0.01 0.32 0.03 flavan3ols 1.31 0.00 1.37 0.01 1.03 0.04 0.80 0.02* 1.07 0.02 1.01 0.07 HCAD, hydroxycinnamic acid derivatives. a Asterisks denote significant differences according to the Student’s test of p<0.05 between non-micro-oxygenated control (NMC) and micro-oxygenated (M) wines. M.J. Cejudo-Bastante et al. / LWT - Food Science and Technology 44 (2011) 866e874 871 terpenes concentration decreased, according to Rapp and Mandery (1986), as well as acetates and short-chain esters (Ramey & Ough, 1980), due to several hydrolysis reactions. With regard to the micro-oxygenation treatment effect, according to the Student’sttest applied, acetaldehyde concentration was increased in micro-oxygenated red wines after five months of storage, thus supporting the above discussion about its implication in the increase of anthocyanin-derived red wine pigments content. Apart from diethyl monosuccinate, the majority ester identified, virtually all of succinic acid derivatives and longchain esters (e.g. ethyl decanoate) had a significantly higher concentration in micro-oxygenated red wines. However, a lower concentration of short-chain esters and acetates was observed (e.g. ethyl butanoate and isoamyl acetate, respectively) after storage. Also, a significant low concentration of C 6 -alcohols in microoxygenated red wines was observed after five months of storage, above all the (E) and (Z) isomers of 3-hexen-1-ol. Volatile acids such as acetic acid showed low concentrations after micro-oxygenation treatment, obtaining wines with lower volatile acidity (Table 1). Despite of the minimal differences in the terpenes, lactones and benzenic fraction, it is highlighted the significantly higher concentration of nerol and d -nonalactone as a consequence of oxygen addition, in agreement with Hernández-Orte et al. (2009), even after five months of storage. Contrarily, it is worth mentioning the diminution of 2-phenylethyl alcohol in micro-oxygenated Cencibel red wines, which could influence negatively on their roses aroma. Alkylpyrazines and tiazols come from the reaction between amino acids and dicarbonylic compounds as Maillard reaction (Pipris-Nicolau, De Revel, Bertrand, & Maujean, 2000), above all under high temperatures. 2,3,5,6-tetramethylpyrazine and 3-ethyl2,5-dimethylpyrazine, previously identified in model solutions and Chardonnay wines (Comuzzo, Tat, Tonizzo, & Battistutta, 2006), Table 5 Volatile compound concentrations ( m g/L) and standard deviation (n¼2) of non-micro-oxygenated control (NMC) and micro-oxygenated (M) Cencibel red wines: after microoxygenation treatment (MOX) and after five months of storage after malolactic fermentation. Only data with significant differences between samples are shown. After MOX b 5 Months of Storage b NMC M NMC M Aldehydes acetaldehyde 53.7 3.01 48.7 3.76 31.7 0.03 40.4 1.96** Esters ethyl butanoate 123 2.36 130 0.63 120 3.17 106 1.98* isoamyl acetate 825 11.4 851 12.7 586 12.6 547 8.60** pentyl hexanoate 6.63 1.26 5.56 0.40 3.28 0.44 6.74 0.03* ethyl decanoate 200 1.77 252 44.0 261 6.59 224 13.1** ethyl methyl succinate 2.87 0.33 3.48 0.57 14.9 0.47 17.9 0.59* 3-methyl butyl octanoate 8.93 0.53 12.4 0.61* 11.3 0.25 8.98 1.65 diethyl succinate a 0.66 0.00 0.76 0.01* 5.95 0.00 6.31 0.05 succinic ester II 2.55 0.08 2.37 0.48 17.6 0.30 21.2 0.70* ethyl propyl succinate 3.16 0.31 2.74 0.26 8.73 0.08 10.0 0.10* 2-phenyl ethyl acetate 78.4 0.91 82.8 1.40* 56.3 0.69 54.5 1.92 succinic ester III 1.50 0.04 1.65 0.09 16.9 0.24 22.1 0.05* ethyl succinate 17.8 0.04 19.0 0.71 24.7 0.15 27.3 0.13* diethyl monosuccinate a nd nd 11.3 0.36 9.90 0.27* ethyl octadecanoate 130 6.49 128 4.48 150 13.6 88.2 6.24* ethyl linoleate 145 3.26 142 24.2 170 5.98 137 2.47* Alcohols 1-pentanol 29.3 4.34 30.5 0.72 34.3 0.94 31.8 0.67** 3-ethoxy1propanol 3.06 0.45 4.77 0.13* 3.36 0.30 2.74 0.28 1-heptanol 11.9 0.14 10.9 0.00* 11.4 0.51 10.4 0.86 1-octanol 10.4 0.28 11.1 0.19* 9.32 0.58 10.0 0.58 Alcohols C6 (E)-3-hexen1ol 12.0 1.00 15.5 0.51* 16.3 2.00 10.8 1.72* (Z)-3-hexen1ol 45.9 0.12 49.4 0.47* 46.5 0.44 43.6 0.77** sum of alcohols C 6 317 3.25 328 0.59* 330.2 9.16 302 2.84** Acids acetic acid 0.59 0.02 0.35 0.02* 0.83 0.10 0.85 0.04 pentanoic acid 2.49 0.07 1.27 0.04* 2.77 0.09 2.76 0.10 (E)-3-hexenoic acid 10.7 0.23 11.3 0.65 11.9 0.26 5.68 0.30* sum of acids a 1.74 0.03 1.75 0.05 1.95 0.03 1.82 0.02* Terpenes nerol 15.0 0.31 18.4 0.27* 19.2 2.05 23.4 1.61 sum of terpenes 149 1.70 150.1 2.99 107 1.03 103 0.88* Benzenic compounds benzyl alcohol 72.3 2.33 70.5 0.49 81.9 0.06 87.0 1.23** phenylethylalcohol a 5.97 0.09 5.44 0.04* 5.96 0.19 5.35 0.09** Lactones d -nonalactone 9.95 1.19 14.3 0.72* 12.1 2.30 12.6 0.54 Pyrazines 3-ethyl2,5dimethylpyrazine 29.0 0.54 30.9 0.42* 36.3 0.39 35.7 0.16 2,3,5,6-tetramethylpyrazine 8.09 0.10 8.54 0.40 6.30 0.86 8.75 0.68** Miscellaneous 4-methyl5tiazolethanol 35.5 0.52 42.8 8.47 99.9 16.1 37.7 0.96** a mg/L. nd, not detected. b Single-asterisks (*) denote significance of p<0.05 for non-micro-oxygenated control (NMC) and micro-oxygenated (M) wines. Double-asterisks (**) denote significance of 0.1 >p>0.05 according to the Student’s test. M.J. Cejudo-Bastante et al. / LWT - Food Science and Technology 44 (2011) 866e874872 showed higher concentration in micro-oxygenated red wines, even after storage conditions. These alkylpyrazines present an herbaceous, cooked potato and burnt aroma, whereas Elisea (2004) described chocolate and nutty aromas for these compounds. However, with regard to 4-methyl-5-tiazolethanol, a concentration decrease was observed. Metallic and slightly nutty notes were described for 4-methyl-5-tiazolethanol by Rowe (2005). 3.5. Micro-oxygenation effect on sensorial properties It stressed out the red fruits notes as the most remarkably sensorial attribute of Cencibel red wines (Fig. 2), and they were significantly lower in micro-oxygenated red wines, according to the Student’sttest applied to the complete set of data. This fact could be probably due to the lower short-chain ethyl ester concentration presented in these wines. On the one hand, opposite to non-micro-oxygenated control red wines, virtually all olfatory main scores increased in microoxygenated red wines as a consequence of the storage period (Fig. 2). With regard to the differences related to oxygen addition, liquorice and spicy values were significantly higher in microoxygenated red wines, contrary to red fruits attribute. This fact could be closely related to the higher concentration of 4-vinylguaiacol and eugenol in these wines, but not statistically significant. These results are in agreement with that Llaudy et al. (2006) found, who observed a significantly higher spicy value in microoxygenated Cabernet Sauvignon red wines, and with Cabanillas, Canals, Rozès, Arola, and Zamora (2001), who described a slightly decrease in red fruits values in a micro-oxygenated coupage of different grape varieties. It is worth to mention that the plum/ currant aroma was only appreciated in micro-oxygenated red wines. According to Pons, Lavigne, Eric, Darriet, and Dubourdieu (2008), this fact could be closely related to b -damascenone and g -nonalactone concentration, by comparison with plum and dry fruits extracts by Gas Cromatography-Olfatometry. On the other hand, contrary to non-micro-oxygenated control red wines, several gustatory descriptors (green taste, acidity and bitterness) suffered a high decrease in micro-oxygenated red wines as a consequence of the storage period (Fig. 2). This fact could be due to the significant lower concentration of C 6 alcohols as a consequence of the micro-oxygenated wines storage,according to López, Ortín, Pérez-Trujillo, Cacho, and Ferreira (2003). Astringency score increased due to the oxygen addition, in agreement with the higher percentage of polymerization, as it had been reported by Pour-Nikfardjam and Kykes (2002). Finally, persistence and global qualities were significant more appreciated for Cencibel microoxygenated red wines. This fact could be due to a distinct character of liquorice, spicy notes and body, as well as the lower green taste, acidity and bitterness values. 4. Conclusions Colour stabilization was observed as a consequence of oxygen addition before malolactic fermentation to Cencibel red wines, 0 1 2 3 4 5 6 7 8 9 10 Red fruits Plum/currant Spicy Liquorice Fresh A A A A Ba b a ba b Olfative B 0 1 2 3 4 5 6 7 8 9 10 Green taste Acidity Bitterness Body Astringency Persistence Qualit y Global Quality a b A B A B A B A B a b a b Gustative Fig. 2. Olfative and gustative attributes mean scores of non-micro-oxygenated control (NMC) and micro-oxygenated (M) Cencibel red wines after micro-oxygenation treatment (MOX) and after five months of storage (5M). Upper-case letters indicate significance of p<0.05 for non-micro-oxygenated control (NMC) and micro-oxygenated (M) wines. Lowercase letters indicate significance of 0.1>p>0.05 according to the Student’s t test. Legend: (-) MOX-NMC; (-) MOX-M; ( ) 5M-NMC; (,) 5M-M. M.J. Cejudo-Bastante et al. / LWT - Food Science and Technology 44 (2011) 866e874 873 even after five months of storage. This fact could be due to the higher concentrations of malvidin-flavan-3-ol mediated by acetaldehyde and pyranoanthocyanins (B-type vitisins), among others. Consequently, Cencibel micro-oxygenated red wines showed a significant higher orange hues (b*), above all after storage conditions. From the volatile compounds point of view, succinic derivatives and long-chain esters concentration was higher in micro-oxygenated red wines after storage, contrarily to the shortchain esters, acetates and C 6 -alcohols. Finally, micro-oxygenation treatment improved the complexity and global quality to Cencibel red wines (plum/currant), increasing the spicy and liquorice values and decreasing herbaceous character after storage conditions. 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