THE EXTRACTION KINETICS OF ANTHOCYANINS AND PROANTHOCYANIDINS FROM GRAPE TO WINE IN THREE DIFFERENT VARIETIES

THE EXTRACTION KINETICS OF ANTHOCYANINS AND PROANTHOCYANIDINS FROM GRAPE TO WINE IN THREE DIFFERENT VARIETIES An B. BAUTISTA-ORTÍN 1, Nir BUSSE-VALVERDE 1, José I. FERNÁNDEZ-FERNÁNDEZ 2, Encrn GÓMEZ-PLAZA 1, Rocío GIL-MUÑOZ 2* 1: Food Science nd Technology Deprtment, Fculty of Veterinry Science, University of Murci, Cmpus de Espinrdo, 30071 Murci, Spin 2: Instituto Murcino de Investigción y Desrrollo Agrrio y Alimentrio, Ctr. L Alerc s/n, 30150 Murci, Spin Astrct Aims: To study the influence of the grpe vriety (Syrh, Monstrell nd Cernet Suvignon) on the extrction kinetics of phenolic compounds (pronthocynidins (PAs) nd nthocynins) during skin mcertion period of 20 dys. Methods nd results: Anthocynins nd PAs were nlyzed y HPLC in three wine vrieties during mcertion time (20 dys). The results showed tht nthocynin extrction followed the sme kinetics for the three vrieties. In the cse of PAs, Syrh must-wine showed the highest concentrtion of these compounds, which were seen to e minly skin-derived. Monstrell must-wine presented the lowest concentrtion of totl PAs nd the lowest percentge of skin-derived PAs. Cernet Suvignon must-wine otined intermedite vlues for totl PAs, lthough these compounds were more polymerized. Conclusion: Grpe vriety nd extrctility ply very importnt role in the wine PA profile. Thus, the highest vlues of totl PAs were oserved in the wines of Cernet Suvignon nd especilly Syrh. Significnt nd impct of the study: Knowledge of the kinetics of ech vriety during the mcertion step my help mnge the composition of wines in light of consumer preference. Key words: nthocynin, pronthocynidin, grpe, wine, kinetic Résumé Ojectifs : Etudier l influence de l vriété de risin (Syrh, Monstrell et Cernet Suvignon) sur l cinétique d extrction des composés phénoliques (nthocynes et pronthocynidines (PAs)) pendnt une période de mcértion de l peu de 20 jours. Méthodes et résultts : Les nthocynes et les PAs ont été nlysées pr HPLC dns trois vriétés de vins pendnt le temps de mcértion (20 jours). Les résultts ont montré que l extrction des nthocynes suivi l même cinétique pour les trois vriétés. Pour les PAs, le moût-vin de Syrh montré l plus forte concentrtion de ces composés, qui provenient principlement de l peu. Le moût-vin de Monstrell présenté l plus file concentrtion de PAs totles et le plus file pourcentge de PAs provennt de l peu. Le moût-vin de Cernet Suvignon otenu des vleurs intermédiires pour les PAs totles, mis ces composés étient plus polymérisés. Conclusion : L vriété de risin et l extrctiilité jouent un rôle très importnt dns le profil pronthocynidinic des vins. Les vleurs les plus élevées de PAs totles ont été oservées dns les vins de Cernet Suvignon et, en prticulier, dns les vins de Syrh. Importnce et impct de l étude : L connissnce de l cinétique de chque vriété lors de l étpe de mcértion peut ider à gérer l composition des vins à l lumière de l préférence des consommteurs. Mots clés: nthocyne, pronthocynidine, risin, vin, cinétique mnuscript received 7th Octoer 2015- revised mnuscript received 27th April 2016 *Corresponding uthor : mrir.gil2@crm.es - 91 - Vigne et Vin Pulictions Interntionles (Bordeux, Frnce)

An B. BAUTISTA-ORTÍN et l. INTRODUCTION Phenolics, minly nthocynins nd pronthocynidins (PAs), re importnt compounds in red wine qulity ecuse they influence colour, mouthfeel nd ging ility. Studies hve shown tht overll intensity nd persistence re positively correlted with stringency, nd therefore to PA content (Mercurio et l., 2010). An optiml extrction of phenolic compounds from the skin nd seeds of grpe erries is crucil to ensure colour stiliztion in the resulting wines nd to imprt desirle mouthfeel properties. The diffusion of the different polyphenols from grpe to must-wine nd, consequently, their extrctility nd finl concentrtion t the end of the mcertion process lrgely depends on their loction in the erry nd the chrcteristics of the different grpe vrieties, especilly those relted to the concentrtion in phenolic compounds nd the extrctility of these polyphenols. Anthocynins re locted in the skins, in the upper cellulr lyers of the hypodermis, while PAs re found in oth skins nd seeds. Skin PAs re minly locted in the skin cell vcuoles (Amrni-Joutei et l., 1994), while seed PAs re found in the epidermis, the outer integument nd the inner integument (Cdot et l., 2006). PAs with upper dihydroxylted unit re clled procynidins nd re present in grpe skin nd seeds, nd PAs with upper trihydroxylted unit re clled prodelphinidins nd re present only in grpe skin (Prieur et l., 1994; Souquet et l., 1996). Grpe skin PAs hve higher men degree of polymeriztion (mdp) nd lower proportion of glloylted suunits thn seed PAs. Both the quntity nd the extrctility of nthocynins nd tnnins increse throughout the grpe ripening. The tnnins nd nthocynins form different complexes with the cell wll components during erry development (Geny et l., 2003). As the erry ripens these complexes re roken up more esily thn in unripe erries. During the mcertion step of the winemking process, phenolic compounds re extrcted from grpes into wine. Anthocynins re minly extrcted in the first dys of fermenttive mcertion (González-Neves et l., 2008), nd most skin PAs, due to their locliztion, re lso soluilized, together with nthocynins. However, the extrction of seed PAs tkes longer. Seed PAs re diffused more slowly nd require longer mcertion times, while their extrction is fvoured y the presence of ethnol (Cnls et l., 2005; González- Mnzno et l., 2004; Del Lludy et l., 2008). This different extrction dynmics of PAs from grpe into wine is therefore n importnt spect to e considered when mnging the mcertion time (Singleton nd Drper, 1964; Boulton, 1995), lthough it my lso e influenced y the grpe vriety nd the technologicl process pplied t the winery, e.g., the extent of erry crushing, pectinolytic enzyme ddition, the use of dry ice or the ppliction of low prefermenttive temperture (Cnls et l., 2008; Cerp-Clderón nd Kennedy, 2008; Busse-Vlverde et l., 2011; Hernández-Jiménez et l., 2012; Butist-Ortín et l., 2013). Grpe vriety is one of the fctors tht hve n importnt influence on the phenolic (PAs nd nthocynins) concentrtion nd composition of the wine. Gonzlez-Neves et l. (2008) studied the influence of grpe vriety on the extrction of nthocynins during fermenttion on skins in three vrieties (Cernet Suvignon, Merlot nd Tnnt) nd concluded tht the nthocynin fingerprint of the young wines otined in clssicl fermenttion is chrcteristic of ech vriety, lthough their initil evolution follows generl tendencies. In the cse of PAs, Busse-Vlverde et l. (2010) reported differences in the PA composition of Monstrell, Syrh nd Cernet Suvignon wines tht were more relted to the vriety thn the winemking technique. A more recent study of these uthors (Busse-Vlverde et l., 2012) with wines of the sme three vrieties nd elorted using different mcertion times gin reported tht the proportion of skin- or seed-derived PAs in wine nd the extrction percentge clerly depend on the vriety, even more thn on skin contct time. The uthors suggested tht this could e due to the existence of different initil concentrtion of grpe PAs, difference in composition or their different degrees of extrctility, which would e ssocited with the composition of the erry cell wlls of the different vrieties. It hs een shown tht the extrction of skin nd seed PAs is incomplete (Bindon et l., 2010; Busse- Vlverde et l., 2010, 2012). The type of PAs, interctions mong themselves nd with other molecules, their dsorption to cell wlls nd their oxidtion, mong other fctors, could influence their extrction (Bindon et l., 2010; Hnlin et l., 2010), nd ll of these my e influenced y vriety. The ojective of this study ws to determine how the grpe vriety ffects the extent of skin nd seed phenolic diffusion from grpes of Monstrell, Cernet Suvignon nd Syrh to wine during 20 dys of fermenttive mcertion nd the finl qulittive nd quntittive composition of these wines t the moment of pressing. Vigne et Vin Pulictions Interntionles (Bordeux, Frnce) - 92 -

MATERIALS AND METHODS Grpes from Vitis vinifer L. cvs. Monstrell (lso known s Mourvèdre), Cernet Suvignon nd Syrh were hrvested in 2010 from commercil vineyrd in Jumill (SE Spin). Grpes were crefully hrvested into 20-kg oxes nd trnsported to the winery. 1. Physicochemicl determintions in grpes Grpe nlysis involved the trditionl flesh mesurements. Totl solule solids (ºBrix) were mesured using digitl refrctometer (Atgo RX- 5000). Titrtle cidity nd ph were mesured using n utomtic titrtor (Metrohm, Herisu, Switzerlnd) with 0.1 N NOH. The methodology for crrying out these nlyses is descried in EEC regultion no. 2676/90. 2. Vinifictions All the vinifictions were mde in triplicte in 100-L stinless steel tnks using 90 kg of grpes. Before lcoholic fermenttion strted, totl cidity ws corrected to 5.5 g/l nd selected yests were dded (Levuline GALA, Oenofrnce, Frnce, 10 g of dry yest/100 kg of grpes). The fermenttive pomce contct period ws 20 dys. All the vinifictions were conducted t 25±1 C. Throughout the fermenttion pomce contct period, the cp ws punched down twice dy nd the temperture nd must density were recorded. At the end of this period, the wines were pressed t 1.5 rs in 75-L tnk memrne press. Free-run nd press wines were comined nd stored t room temperture, the cp ws punched down twice dy nd the temperture nd must density were recorded. The nlyses were crried out every 2 dys during the mcertion period. 3. Determintion of nthocynins in grpes nd wines Grpes were peeled with sclpel nd the skins nd seeds were stored t -20 C until nlysis. Smples (2 g) were immersed in methnol (40 ml) in hermeticlly closed tues nd plced on stirring plte t 150 rpm nd 25 C. After two hours, the methnolic extrcts were filtered through 0.45-µm memrne nd nlyzed y high-performnce liquid chromtogrphy (HPLC). Smples of wine were similrly filtered nd directly nlyzed y HPLC ccording to Butist-Ortín et l. (2005). 4. Determintion of pronthocynidins in grpes nd wines The seeds nd skins of 10 erries were seprted from the mesocrp nd rinsed with distilleddeionized wter. Whole seeds nd skins were extrcted seprtely in covered Erlenmeyer flsks with 10 ml of 2:1 cetone/wter t room temperture for 24 h on n oritl shker t 200 rpm. To minimize PA oxidtion, solutions were spurged with nitrogen nd the extrction ws crried out in the drk. Following extrction, the extrct ws concentrted under reduced pressure t 35 ºC to remove cetone, nd then lyophilized to dry powder. This powder ws redissolved in 1 ml methnol in volumetric flsk. Skin nd seed PAs were determined ccording to the method descried y Kennedy nd Jones (2001) with some modifictions, s follows. A solution of 0.2 N HCl in methnol, contining 100 g/l phloroglucinol nd 20 g/l scoric cid ws prepred (phloroglucinolysis regent). The methnolic extrct ws rected with the phloroglucinolysis regent (1:1) in wter th for 20 minutes t 50 ºC nd then comined with 2 volumes of 200 mm queous sodium cette to stop the rection. Blnks from grpe seeds nd skins were nlyzed using the methnolic extrcts without the phloroglucinolysis regent in order to not infr-estimte the mdp nd the totl PA content. For wines, the smples were prepred y n optimiztion of the method descried y Pstor del Río nd Kennedy (2006). For this, 5 ml of wine ws evported in CentriVp concentrtor (Lconco, USA), redissolved in 3 ml wter nd then pssed through C18-SPE column (1 g, Wters, Milford, USA), previously ctivted with 10 ml methnol followed y 20 ml wter. The crtridge ws wshed with 20 ml wter, nd the compounds of interest were eluted with 10 ml methnol, evported, nd then dissolved in 1 ml methnol. Phloroglucinolysis ws then crried out s descried ove. HPLC nlysis followed the conditions descried y Busse-Vlverde et l. (2010). PA clevge products were estimted using their response fctors reltive to (+)-ctechin, which ws used s the quntittive stndrd. These nlyses llowed the totl PA content, the pprent mdp nd the percentge of ech constitutive unit to e determined. The mdp ws clculted s the sum of ll suunits (flvn-3-ol monomer nd phloroglucinol dducts, in moles) divided y the sum of ll flvn-3-ol monomers (in moles). - 93 - Vigne et Vin Pulictions Interntionles (Bordeux, Frnce)

An B. BAUTISTA-ORTÍN et l. Totl tnnins (mg/kg) 1800 1600 1400 1200 1000 800 600 400 Monstrell C.Suvignon Syrh A mdp 25 20 15 10 5 Monstrell C.Suvignon Syrh B 200 0 0 Skins Seeds Skins Seeds % Glloyltion 20 18 16 14 12 10 8 Monstrell C.Suvignon Syrh C % EGct 60 50 40 30 c Monstrell C.Suvignon Syrh D 6 4 2 20 10 0 0 Skins Seeds %EGCt skins %EGCt seeds Figure 1. Men concentrtion nd composition vlues of skin nd seed pronthocynidins in erries of the three studied vrieties. The PAs extrcted from skin nd seeds during winemking were determined following the method proposed y Peyrot des Gchons nd Kennedy (2003). 5. Sttisticl dt tretment Significnt differences mong smples nd for ech vrile were ssessed y nlysis of vrince (ANOVA). A Duncn test ws used to seprte the mens (p<0.05) when the ANOVA test ws significnt, using the sttisticl pckge Sttgrphics 5.0 Plus. RESULTS AND DISCUSSION 1. Physicochemicl determintions in grpes The physicochemicl dt of grpes t the moment of hrvest re shown in Tle 1. Similr results were found in ph nd cidity prmeters (mg/l). But in contrst, sttisticl differences were found in ºBrix, indicting tht the Monstrell grpes were the lest mture nd Syrh grpes the most mture. Finlly, the size of the erries ws different mong the three vrieties studied; Cernet Suvignon grpes showed the smllest erries nd Monstrell grpes the iggest erries. These differences cn influence in the mcertion process, esides grpe vriety. 2. Phenolic profile of grpe for the three vrieties Tle 2 shows the nthocynin composition of the grpes of the three vrieties studied t the time of hrvest nd the reltive percentge of the different kinds of compounds. When the results were expressed s µg/g of skin, the highest vlues were oserved in Syrh grpes, intermedite vlues in Monstrell grpes nd lowest vlues in Cernet Suvignon grpes. If the results re expressed s mg/kg of erry, totl nthocynins were lso higher in Syrh grpes (1555 mg/kg erry), followed y Cernet Suvignon (946 mg/kg erry) nd then Monstrell (938 mg/kg erry). Romero-Cscles et l. (2005) found the highest nthocynin vlues expressed s µg/g of skin or mg/kg of erry in Monstrell nd Syrh grpes, while Cernet Suvignon grpes hd the lowest concentrtion. Tle 1. Physicochemicl chrcteristics in grpes t hrvest Weight 100 g ph Acidity ºBrix Monstrell 122.02 ± 1.37 3.57 ± 0.07 5.26 ± 0.01 24.2 ± 0.05 C. Suvignon 80.39 ± 3.12 3.66 ± 0.05 5.03 ± 0.04 24.7 ± 0.06 Syrh 105.24 ± 2.45 3.57 ± 0.09 5.41 ± 0.17 25.1 ± 0.10 Vigne et Vin Pulictions Interntionles (Bordeux, Frnce) - 94 -

Tle 2. Concentrtion of nthocynins in erries of the three vrieties t hrvest Totl nthocynins Totl nthocynins ( g/g skin) (mg/kg erry) % Non cylted % Acylted % Dihydroxylted % Trihydroxylted Monstrell 10611.8 1 937.6 64.8 c 35.1 30.7 c 69.2 C. Suvignon 8882.0 945.9 43.7 56.3 10.7 89.3 Syrh 18796.9 c 1554.6 31.4 68.6 c 7.0 93.0 c 1 Different letters in the sme row indicte different levels of significnce ccording to Duncn test (p<0.05). Totl Anthocynins (mg/l) The highest non-cylted nthocynin percentge nd the lowest proportion of cylted nthocynins were found in Monstrell grpes. A similr result ws lso oserved y Romero-Cscles et l. (2005) nd Grcí-Beneytez et l. (2002). Syrh nd Cernet Suvignon grpes hd higher percentges of cylted nthocynins (69 nd 56%, respectively). For the three vrieties, trihydroxylted monoglucosides were present in higher proportions thn dihydroxylted monoglucosides lthough the highest percentge of the ltter ws oserved in Monstrell grpes, in which the vlues were sttisticlly different from those otined for Syrh nd Cernet Suvignon. It hs een stted tht grpes in which the trihydroxylted nthocynins (delphinidin, petunidin nd mlvidin) re predominnt re much more pigmented nd provide wines with more stle colour (Fernández- López et l., 1998; Romero-Cscles et l., 2005). Figure 1 shows the totl PA content (mg/kg erry), mdp of PAs, glloyltion percentge nd the percentge of extension (-)-epiglloctechin for the three vrieties studied t the time of hrvest. As regrds skins, the highest PA content corresponded to Cernet Suvignon grpes, the sme eing true in the cse of seeds, lthough no sttisticl differences were found mong vrieties. The mdp ws lwys 1600 1400 1200 1000 800 600 400 200 0 2 4 6 8 10 12 14 16 18 20 Mcertion dys Monstrell Cernet Suvignon Syrh Figure 2. Evolution of totl nthocynins during the mcertion period of the three studied vrieties. higher in skin thn seed PAs, the most polymerized PAs eing those of Cernet Suvignon grpe skins nd Monstrell grpe seeds. The glloyltion percentge ws higher in seed PAs thn in those from skin. Cernet Suvignon grpes showed the lowest levels of tnnins to sttisticlly significnt extent. Cernet Suvignon grpes presented the highest content of prodelphinidins, while Monstrell grpes showed the lowest content of these compounds. It seems tht the iosynthesis of flvnols nd PAs in the grpe is highly regulted t the vriety level, which leds to differences tht could ply significnt role in the technologicl nd sensory properties of the resulting wines (Mttivi et l., 2009). 3. Totl nthocynins extrcted during the mcertion period The totl nthocynin concentrtion ws nlyzed every two dys during the mcertion period, which lsted 20 dys (Figure 2). During this time, nthocynin extrction followed similr kinetics in ll three vrieties, the concentrtion incresing from the eginning of mcertion until reching mximum nd then decresing slowly until the end of the process. The min differences for the three vrieties were found during the first dys of mcertion. The nthocynin concentrtion incresed rpidly in Syrh nd Cernet Suvignon mustwines, reching the highest concentrtion t dy 6 (1488 mg/l nd 1085 mg/l, respectively). In Monstrell grpes, nthocynin extrction into the wine ws slower, reching mximum vlue t dy 8, lthough lower thn in the other vrieties (783 mg/l). The extrction of nthocynins from grpe skins ws clerly incomplete. So, considering our winemking conditions, the rel percentge of extrction of skin nthocynins t the moment of mximum extrction in the wines ws 80% for Cernet Suvignon grpes, 67% for Syrh grpes nd 58% for Monstrell grpes. It hs een suggested tht n equilirium sed on dsorption-desorption is estlished etween the nthocynin concentrtion of the grpe nd the wine nd, when this equilirium - 95 - Vigne et Vin Pulictions Interntionles (Bordeux, Frnce)

An B. BAUTISTA-ORTÍN et l. hs een reched, no more nthocynins cn e extrcted from grpe skins into the wine (Boulton, 2001). The lower extrction of nthocynins from Monstrell grpes compred with Syrh nd Cernet Suvignon grpes cn e ttriuted to the cell wll composition nd the more rigid structure of this vriety (Romero-Cscles et l., 2005; Orteg- Regules et l., 2008). After, nthocynin levels decresed until the end of mcertion, t which time the Syrh nd Cernet Suvignon wines showed close vlues, while Monstrell wines continued to show the lowest nthocynin content. A similr result to tht otined in this study with Monstrell vriety ws lso found y Butist-Ortín et l. (2004), lthough these uthors pointed to more pronounced decrese in the nthocynin content fter the mximum vlue hd een reched. The nthocynin composition of the musts my e modified significntly y the loss of n importnt frction of these molecules through dsorption of the yests. These nthocynins precipitte long with the ded yests once fermenttion hs finished, ecoming prt of the lees (Vsserot et l., 1997; Mort et l., 2003). Other cuses cn e the refixtion on the skins, the fixtion on the seeds, nd the rections of the oxidtion, condenstion nd cycloddition in which nthocynins re involved. In our cse, the three vrieties otined similr lcohol content nd totl cidity t the end of lcoholic fermenttion (dt not shown), so these prmeters did not ffect to extrction kinetics. 4. Pronthocynidin extrction during mcertion Figures 3 nd 4 show the concentrtion of totl PAs, mdp, the concentrtion of skin-seed derived PAs, glloyltion percentge nd percentge of extension epiglloctechin during the 20 dys of fermenttive mcertion for the three vrieties. Smples were nlyzed every two dys. Tnnin extrction cn e descried s diffusion process (Boulton, 1995), lthough skin nd seed tnnins hve different extrction kinetics. Most of the tnnins in the skins, due to their loction, egin to e soluilized together with nthocynins, lthough removl is slow process. In contrst, the seeds hve slow diffusion rte, which is ccelerted to hlf of fermenttion, when lcohol fcilittes dissolution of the cuticle. In our study, the extrction kinetics of totl PAs ws rpid during the first few dys of mcertion; it then stilized with slow extrction kinetics (s in the cse of Monstrell vriety), efore decresing until the end of the process. After two Totl PAs (mg/l) Skin PAs (mg/l) Seed PAs (mg/l) dys of mcertion, no differences were oserved in the PA content mong the must-wines of the three vrieties, lthough differences ecme more evident s mcertion time progressed, perhps due to the different diffusion rtes of the PAs in the different vrieties. This diffusion rte ws highest for the PAs of Syrh, followed y those of Cernet Suvignon nd Monstrell. The highest totl PA extrction rte ws reched on dy 10 for Cernet Suvignon wine (950 mg/l), dy 12 for Syrh wine (1053 mg/l) nd dy 18 for Monstrell wine (649 mg/l). The vlues then remined stle in Cernet Suvignon nd Syrh wines, lthough ner the end of the mcertion period, the totl PA concentrtion diminished slightly. 1200 1000 800 600 400 200 900 800 700 600 500 400 300 200 100 300 250 200 150 100 50 A B C Mcertion dys Monstrell C. Suvignon Syrh 2 4 6 8 10 12 14 16 18 20 Figure 3. Pronthocynidin concentrtion during mcertion period. Vigne et Vin Pulictions Interntionles (Bordeux, Frnce) - 96 -

DPm %Glloyltion %Epiglloctechin 9 6 3 5 4 3 2 1 A B 40 C 30 20 10 Monstrell C. Suvignon Syrh 2 4 6 8 10 12 14 16 18 20 Mcertion dys Figure 4. Pronthocynin composition concentrtion during mcertion period. Throughout the mcertion period, the Syrh wine presented the highest totl PA content, lthough the Syrh grpes hd shown the lowest skin PA content nd similr seed PA content to the other vrieties. The Monstrell grpes produced wines with the lowest PA content, while Cernet Suvignon grpes gve wines with intermedite vlues. The PA content found in wines represents only prt of the PAs present in grpes nd, lthough this content increses in wine with mcertion time, it lso depends on the vriety, since the Cernet Suvignon grpe, which presented the highest PA content, did not produce the wine with the highest PA concentrtion. It ppers tht the extrction of tnnins is highly vrile, depending on the vriety, so tht mesurement of the totl tnnin content in grpe my not lwys e useful for predicting the tnnin content of the resulting wine, s lso oserved y Adms nd Scholz (2009) nd Butist-Ortín et l. (2013). Romero-Cscles et l. (2005) lso demonstrted tht Syrh grpes, which showed lower skin PA vlue thn Monstrell grpes nd similr seed PA vlue, produced wine with similr PA content to tht of Monstrell. These uthors concluded tht the content of PAs in wine did not correlte with the content of these compounds in grpes ut did so with phenolic extrctility. This prmeter, totl PA content, ppers to e closely relted to the different composition of the cell wlls in the different vrieties (Orteg-Regules et l., 2006). Tking into ccount the PA composition mesured in grpes nd following the method descried y Peyrot des Gchons nd Kennedy (2003), it ws possile to determine the skin nd seed contriution to the PA composition of must-wine (Figure 3, B nd C). The extrction of skin PAs during mcertion lso presented very different extrction kinetics for the three vrieties, ut quite similr to tht oserved in the cse of totl PAs. The highest skin extrction vlues were otined for Syrh wines, followed y Cernet Suvignon wines nd, lstly, Monstrell wines. Mongs et l. (2003) lso indicted tht wine PAs hd more similr profile to skin PAs thn those of seeds. In Syrh wines, skin PA extrction reched mximum t dy 8 nd in Cernet Suvignon must-wines t dy 10, nd, in oth wines, fter plteu, the concentrtion decresed slightly until the end of the mcertion period. In Merlot grpes, Cerp-Clderón nd Kennedy (2008) found the mximum proportion of PAs extrcted from skin on the 9th dy of mcertion, followed y stiliztion of the concentrtion until dy 18, fter which no further extrction occurred. These uthors suggested tht this my reflect the continued dsorption of extrcted PAs to suspended cell wll mteril nd, possily, yest cells. In the cse of our Monstrell wines, the extrction of skin PAs continued until dy 18, just two dys efore mcertion finished, in process tht ws slower nd in which the quntity extrcted ws lower thn for the other vrieties. The lower extrction in this vriety my e relted to the greter mount of cell wll mteril nd thicker wlls, s found y Orteg-Regules et l. (2008), in Monstrell compred with Syrh nd Cernet Suvignon, which my ct s stronger rrier to the extrction of PAs locted inside skin cells. As regrds seed-derived PAs, the extrction kinetics ws similr for the three vrieties; their extrction ws incresing from the eginning to the end of the mcertion period. The mounts otined for the three vrieties were much lower compred with the skin-derived PAs, especilly for Cernet Suvignon - 97 - Vigne et Vin Pulictions Interntionles (Bordeux, Frnce)

An B. BAUTISTA-ORTÍN et l. nd Syrh. In the cse of Monstrell, the vlues of oth skin nd seed PAs were closer. These results gree with those otined y González-Mnzno et l. (2004), Del Lludy et l. (2008) nd Cerp- Clderón nd Kennedy (2008). The mdp of the PAs (Figures 4, A) incresed more quickly for Syrh wine, reching mximum t dy 4, while Cernet Suvignon nd Monstrell reched the mximum vlue two dys lter (dy 6). The vlues of this prmeter then decresed slightly for Monstrell, ut decresed y close to two units for Syrh, efore remining stle until dy 18 of mcertion. In Cernet Suvignon there ws lso slight decrese in mdp vlues, which occurred severl dys lter thn in Monstrell nd Syrh. At the end of the mcertion process, the mdp vlues slightly incresed in ll three vrieties. The highest mdp of PAs during the first dys of mcertion ws reched in Syrh wine followed y Cernet Suvignon wine, while from hlf wy through mcertion the highest mdp vlue ws seen in Cernet Suvignon, the PAs of Syrh wine showing intermedite vlues. This result my e due to the skin PAs of Cernet Suvignon eing more polymerized. Therefore, mdp of the PAs present in wine ws seen to e most ffected during the first few dys of mcertion, which does not coincide with the findings of Del Lludy et l. (2008), who found no mjor chnges in mdp vlues fter ppliction of 25 dys of mcertion. The increse in mdp vlues coincided with n increse in the extrction of skin-derived PAs in the wines. However, during the mcertion period, n increse in the skin-derived PAs ws not lwys reflected in the stte of PA polymeriztion. This my e due to severl cuses: i) the simultneous extrction of seed-derived PAs, which re less polymerized nd my compenste the vlues of mdp, ii) the dsorption of these compounds minly y suspended flesh cellulr wll mteril, for which the very high moleculr weight skin PAs hve high ffinity (Bindon et l., 2010), iii) process of oxidtion (Poncet-Legrnd et l., 2010) nd interction with other phenolic compounds of wine nd iv) the low efficiency of the hydrolcoholic medium (s is the must-wine) in the extrction of high moleculr weight PAs, s previously shown y Butist-Ortín et l. (2013). The glloyltion percentge of the PAs (Figure 4, B) ffects oth the itterness nd stringency of wines (Lesscheve nd Nole, 2005). This prmeter incresed for ll vrieties until dy 18, proly due to the increse in the contriution of seed PAs, which re more glloylted thn those of skin. It then fell slightly until the end of mcertion, proly due to the relese of gllic cid units from their corresponding glloylted precursors (Singleton nd Trousdle, 1983). Although the PAs of Monstrell wine during the first dys of mcertion showed lower glloyltion percentge vlues thn the wines of the other vrieties, this prmeter then incresed very quickly, reching the highest vlue t the moment of pressing. The PAs of Cernet Suvignon wine presented the lowest glloyltion percentge vlues. These results re consistent with those mesured in the grpes. The evolution of the extension (-)-epiglloctechin percentge during mcertion reflects the extrction of prodelphinidins into the wine (Figure 4, C). This prmeter incresed during the first dys of mcertion nd then diminished slightly until the end of the process. This decrese could e relted to the oxidtion of PAs, in which this suunit is present, since it hs een suggested tht the flvn-3-ols trihydroxyls re more susceptile to oxidtion during the fermenttion process thn di-hydroxyls, such s (+)-ctechin nd (-)-epictechin, due to differences in the redox potentil (Nnjo et l., 1996). This would explin the lower proportion of skin PAs in mustwine during mcertion (Aron nd Kennedy, 2007), lthough the contriution of seed PAs, which do not contin (-)-epiglloctechin, my lso contriute to the decrese. The percentge of prodelphinidins differed in the must-wines of the three vrieties. Syrh wine reched the mximum vlue t dy 4 nd Cernet Suvignon nd Monstrell wines four dys lter, Cernet Suvignon must-wines showing the highest vlue of this prmeter nd Monstrell the lowest, which, gin, indictes the difficulty of extrcting the phenolic compounds from the skins of Monstrell grpes. Frncis et l. (2002) reported tht high prodelphinidin content in wine cn decrese the stringency senstion nd soften the mouthfeel of the wine. CONCLUSION It is cler tht grpe vriety nd extrctility ply very importnt role in the wine PA profile. Thus, the highest vlues of totl PAs were oserved in the wines of Cernet Suvignon nd, especilly, Syrh due to greter contriution of PAs from grpe skins. This ws reflected in higher mdp vlues nd higher percentge of (-)-epiglloctechin, together with Vigne et Vin Pulictions Interntionles (Bordeux, Frnce) - 98 -

low glloyltion percentge. In contrst, Monstrell wine presented the lowest PA vlues; PAs were lso less polymerized, ut showed high percentge of glloyltion nd lower percentge of (-)- epiglloctechin due to the greter contriution of seeds compred to skins. Therefore, s regrds the phenolic content nd composition of the resulting wines, it is importnt to know how ech vriety ehves during the mcertion step. This will enle winemkers to choose the most suitle mcertion time ccording to the type of wine desired, since these two prmeters will hve gret influence on the finl orgnoleptic chrcteristics of the wine. Acknowledgements: This work ws mde possile y finncil ssistnce from the Ministerio de Cienci e Innovción, project AGL2009-12503. REFERENCES Adms DO. nd Scholz RC., 2009. Tnnins: the prolem of extrction. In: Proceedings of the 13th Austrlin Wine Industry Technicl Conference, Adelide, pp. 160-164. Amrni Joutei K., Glories Y. nd Mercier M., 1994. Loclistion des tnins dns l pellicule de ie de risin. Vitis, 33, 133-138. Aron PM. nd Kennedy JA., 2007. Compositionl investigtion of phenolic polymers isolted from Vitis vinifer L. cv. Pinot Noir during fermenttion. J. Agric. Food Chem., 55, 5670-5680. Butist-Ortín AB., Fernández-Fernández JI., López-Roc JM. nd Gomez-Plz E., 2004. Wine-mking of high coloured wines: extended pomce contct nd run-off of juice prior to fermenttion. Food Sci. Technol. Int., 10, 287-295. Butist-Ortín AB., Mrtínez-Cutills A., Ros-Grcí JM., López-Roc JM. nd Gómez-Plz E., 2005. Improving colour extrction nd stility in red wines: the use of mcertion enzymes nd enologicl tnnins. Int. J. Food Sci. Technol., 40, 867-878. Butist-Ortín AB., Jiménez-Pscul E., Busse-Vlverde N., López-Roc JM., Ros-Grcí JM. nd Gómez- Plz E., 2013. Effect of wine mcertion enzymes on the extrction of grpe seed pronthocynidins. Food Bioprocess Technol., 6, 2207-2212. Bindon KA., Smith PA. nd Kennedy JA., 2010. Interction etween grpe-derived pronthocynidins nd cell wll mteril. 1. Effect on pronthocynidin composition nd moleculr mss. J. Agric. Food Chem., 58, 2520-2528. Bindon KA., Smith PA., Holt H. nd Kennedy JA., 2010. Interction etween grpe-derived pronthocynidins nd cell wll mteril. 2. Implictions for vinifiction. J. Agric. Food Chem., 58, 10736-10746. Boulton R., 1995. Red wines. In: Fermented Beverge Production, Le AGH. nd Piggott JR. (Eds.), Blckie Acdemic nd Professionl, Chpmn & Hll, London, pp. 121-158. Boulton R., 2001. The copigmenttion of nthocynins nd its role in the color of red wine: criticl review. Am. J. Enol. Vitic., 52, 67-87. Busse-Vlverde N., Gómez-Plz E., López-Roc JM., Gil-Muñoz R., Fernández-Fernández JI. nd Butist-Ortín AB., 2010. Effect of different enologicl prctices on skin nd seed pronthocynidins in three vrietl wines. J. Agric. Food Chem., 58, 11333-11339. Busse-Vlverde N., Gómez-Plz E., López-Roc JM., Gil-Muñoz R. nd Butist-Ortín AB., 2011. The extrction of nthocynins nd pronthocynidins from grpes to wine during fermenttive mcertion is ffected y the enologicl technique. J. Agric. Food Chem., 59, 5450-5455. Busse-Vlverde N., Butist-Ortín AB., Gómez-Plz E., Fernández-Fernández JI. nd Gil-Muñoz R., 2012. Influence of skin mcertion time on the pronthocynidin content of red wines. Eur. Food Res. Technol., 235, 1117-1123. Cdot Y., Minn-Cstello MT. nd Chevlier M., 2006. Antomicl, histologicl, nd histochemicl chnges in grpe seeds from Vitis vinifer L. cv Cernet frnc during fruit development. J. Agric. Food Chem., 54, 9206-9215. Cnls R., Lludy MC., Vlls J., Cnls JM. nd Zmor F., 2005. Influence of ethnol concentrtion on the extrction of color nd phenolic compounds from the skin nd seeds of Temprnillo grpes t different stges of ripening. J. Agric. Food Chem., 53, 4019-4025. Cnls R., Lludy MC., Cnls JM. nd Zmor F., 2008. Influence of the elimintion nd ddition of seeds on the colour, phenolic composition nd stringency of red wine. Eur. Food Res. Technol., 226, 1183-1190. Cerp-Clderón FK. nd Kennedy JA., 2008. Berry integrity nd extrction of skin nd seed pronthocynidins during red wine fermenttion. J. Agric. Food Chem., 56, 9006-9014. Del Lludy MC., Cnls R., Cnls JM. nd Zmor F., 2008. Influence of ripening stge nd mcertion length on the contriution of grpe skins, seeds nd stems to phenolic composition nd stringency in wine-simulted mcertions. Eur. Food Res. Technol., 226, 337-344. Fernández-López JA., Almel L., Muñoz JA., Hidlgo V. nd Crreño J., 1998. Dependence etween colour nd individul nthocynin content in ripening grpes. Food Res. Int., 31, 667-672. Frncis IL., Gwel R., Illnd PG., Vidl S., Cheynier V., Guyot S. nd Kwitkowski MJ., 2002. - 99 - Vigne et Vin Pulictions Interntionles (Bordeux, Frnce)

An B. BAUTISTA-ORTÍN et l. Chrcterising mouth-feel properties of red wines. Aust. NZ. Wine Ind. J., 17, 18-25. Grcí-Beneytez E., Revill E. nd Cello F., 2002. Anthocynin pttern of severl red grpe cultivrs nd wines mde from them. Eur. Food Res. Technol., 215, 32-37. Geny L., Sucier C., Brcco S., Dviud F. nd Glories Y., 2003. Composition nd cellulr locliztion of tnnins in grpe seeds during mturtion. J. Agric. Food Chem., 51, 8051-8054. González-Mnzno S., Rivs-Gonzlo JC. nd Sntos- Buelg C., 2004. Extrction of flvn-3-ols from grpe seed nd skin into wine using simulted mcertion. Anl. Chim. Act, 513, 283-289. González-Neves G., Gil G. nd Brreiro L., 2008. Influence of grpe vriety on the extrction of nthocynins during the fermenttion on skins. Eur. Food Res. Technol., 226, 1349-1355. Hnlin R., Hrmov M., Hrertson JF. nd Downey M., 2010. Review: condensed tnnin nd grpe cell wll interctions nd their impct on tnnin extrctility into wine. Aust. J. Grpe Wine Res., 16, 179-188. Hernández-Jiménez A., Kennedy JA., Butist-Ortín AB. nd Gomez-Plz E., 2012. Effect of ethnol on grpe seed pronthocynidin extrction. Am. J. Enol. Vitic., 63, 57-61. Kennedy JA. nd Jones GP., 2001. Anlysis of pronthocynidin clevge products following cidctlysis in the presence of excess phloroglucinol. J. Agric. Food Chem., 49, 1740-1746. Lesscheve I. nd Nole AC., 2005. Polyphenols: fctors influencing their sensory properties nd their effects on food nd everge preferences. Am. J. Clin. Nutr., 81, 330-335. Mttivi F., Vrhovsek U., Msuero D. nd Trinotti D., 2009. Differences in the mount nd structure of extrctle skin nd seed tnnins mongst red grpe vrieties. Aust. J. Grpe Wine Res., 15, 27-35. Mercurio MD., Dmergs RG., Cozzolino D., Herderich MJ. nd Smith PA., 2010. Reltionship etween red wine grdes nd phenolics: 1. Tnnin nd totl phenolics concentrtions. J. Agric. Food Chem., 58, 12313-12319. Mongs M., Gómez-Cordovés MC., Brtolomé B., Lureno O. nd Ricrdo-D-Silv JM., 2003. Monomeric, oligomeric nd polymeric flvn-3-ol composition of wines nd grpes from Vitis vinifer L. c.v. Grcino, Temprnillo nd Cernet Suvignon. J. Agric. Food Chem., 51, 6475-6481. Mort A., Gómez-Cordovés MC., Colomo B. nd Suárez JA., 2003. Pyruvic cid nd cetldehyde production y different strins of Scchromyces cerevisie: reltionship with vitisin A nd B formtion in red wines. J. Agric. Food Chem., 51, 7402-7409. Nnjo F., Goto K., Seto R., Suzuki M., Ski M. nd Hr Y., 1996. Scvenging effects of te ctechins nd their derivtives on 1,1-diphenyl-2-picrylhydrzyl rdicl. Free Rdic. Biol. Med., 21, 895-902. Orteg-Regules A., Romero-Cscles I., Ros-Grci JM., Lopez-Roc JM. nd Gomez-Plz E., 2006. A first pproch towrds the reltionship etween grpe skin cell-wll composition nd nthocynin extrctility. Anl. Chim. Act, 563, 26-32. Orteg-Regules A., Ros-Grcí JM., Butist-Ortín AB., López-Roc JM. nd Gómez-Plz E., 2008. Differences in morphology nd composition of skin nd pulp cell wlls from grpes (Vitis vinifer L.): technologicl implictions. Eur. Food Res. Technol., 227, 223-231. Pstor del Río JL. nd Kennedy JA., 2006. Development of pronthocynidins in Vitis vinifer L. cv. Pinot noir grpes nd extrction into wine. Am. J. Enol. Vitic., 57, 125-132. Peyrot des Gchons C. nd Kennedy JA., 2003. Direct method for determining seed nd skin pronthocynidin extrction into red wine. J. Agric. Food Chem., 51, 5877-5881. Poncet-Legrnd C., Cne B., Butist-Ortín AB., Crrillo S., Fulcrnd H., Perez J. nd Vernhet A., 2010. Tnnin oxidtion: intr- versus intermoleculr rections. Biomcromolecules, 11, 2376-2386. Prieur C., Rigud J., Cheynier V. nd Moutounet M., 1994. Oligomeric nd polymeric procynidins from grpe seeds (Vitis vinifer). Phytochemistry, 36, 781-784. Romero-Cscles I., Fernández-Fernández JI., López-Roc JM. nd Gómez-Plz E., 2005. The mcertion process during winemking extrction of nthocynins from grpe skins into wine. Eur. Food Res. Technol., 221, 163-167. Singleton VL. nd Drper DE., 1964. The trnsfer of polyphenolic compounds from grpe seeds into wines. Am. J. Enol. Vitic., 15, 34-40. Singleton VL. nd Trousdle E., 1983. White wine phenolics: vrietl nd processing differences s shown y HPLC. Am. J. Enol. Vitic., 34, 27-34. Souquet JM., Cheynier V., Brossud F. nd Moutounet M., 1996. Polymeric pronthocynidins from grpe skins. Phytochemistry, 43, 509-512. Vsserot Y., Cillet S. nd Mujen A., 1997. Study of nthocynin dsorption y yest lees. Effect of some physicochemicl prmeters. Am. J. Enol. Vitic., 48, 433-437. Vigne et Vin Pulictions Interntionles (Bordeux, Frnce) - 100 -