Research Note Relationship between Skin Break Force and Anthocyanin Extractability at Different Ripening Stages

Transcription

1 Reserch Note Reltionship etween Skin Brek Force nd Anthocynin Extrctility t Different Ripening Stges Luc Rolle, 1 * Frizio Torchio, 2 Giuseppe Zepp, 1 nd Vincenzo Geri 3 Astrct: The extrctility of nthocynins from winegrpes with different skin hrdness t two different ripening stges ws evluted. Skin hrdness ws ssessed y texture nlysis, rpid nd low-cost nlyticl technique. Grpe erries of cv. Brchetto were clirted ccording to their density estimted y flottion in 10 different slt solutions. Skin hrdness ws mesured vi puncture test, nd two groups of erries with different skin hrdness were selected for ech ripening stge: soft (<0.40 N) nd hrd (>0.50 N). Anthocynin skin extrction ws evluted in hydrolcoholic model solution nd the kinetics of skin dissolution ws monitored. For ech ripening stge, the hrder skin hd greter nthocynin extrctility. Significnt interctions etween ripening stge nd skin hrdness were found in the composition of individul nthocynins present in the extrct. Key words: extrctility, nthocynin, skin hrdness, texture nlysis, puncture test The visul chrcteristic of red wine is minly due to nthocynins tht re present in the grpe skin t hrvest nd tht dissolve in the red wine during the mcertion/ fermenttion process (Amrni Joutei nd Glories 1995, Romero-Cscles et l. 2005). Anthocynins ccumulte grdully in erry skins during ripening (Fernández-López et l. 1992), nd their totl mount t hrvest depends on vriety (i.e., pink, red, or lck skin vriety) nd mny other fctors, including environmentl prmeters nd gronomic prctice (Guidoni et l. 2008). The types nd the mounts of vrious nthocynins in grpe skins (their profile ) determine the color of resulting wines, ut their extrction from the grpe skin into the wine depends on the tendency of the erry skin to relese them (González-Neves et l. 2004, Romero-Cscles et l. 2005). The extrctility of nthocynins increses throughout grpe ripening s consequence of cell wll degrdtion y pectolytic enzymes (Riéreu-Gyon et l. 2000). Differences in polyscchrides sed on glctose nd rinose, the cellulose content, nd the degree of methyltion of the pectins could e responsile for the difference in nthocynin extrctility (Orteg-Regules et l. 2006, 2008). 1 Resercher, 2 Ph.D. student, 3 Professor, Di.V.P.R.A., Microiology nd Food Technology sector, University of Turin, Vi L. d Vinci 44, Gruglisco, Torino, Itly. *Corresponding uthor (emil: luc.rolle@unito.it; tel: ; fx: ) Acknowledgment: This study ws funded y Consorzio Tutel Vini d Acqui, Brchetto d Acqui. Mnuscript sumitted My 2008, revised July 2008, ccepted Sept Puliction costs of this rticle defryed in prt y pge fees. Copyright 2009 y the Americn Society for Enology nd Viticulture. All rights reserved. 93 Severl studies hve ttempted to define the est method to evlute polyphenolic compounds in grpes nd their ese of relese. These nlyticl methods re generlly rther complex nd often require long nlysis times (Cgnsso et l. 2008). Furthermore, some dt interprettion my e difficult (Venencie et l. 1998). Texture nlysis is current nlyticl technique used for mesurement of the physicl properties of food. For winegrpes, the literture contins studies on modifictions of some grpe texturl properties during ripeness (Al et l. 1992, Roin et l. 1997, Lee nd Bourne 1980, Grotte et l. 2001) nd the influence of climte nd growing loction on mechnicl ehvior (Le Moigne et l. 2008). The im of this work ws to exmine the reltionship etween skin hrdness, determined y puncture test, nd the ese of extrctility of nthocynins in erries t different ripening stges. The study ws crried out on Brchetto, n importnt romtic red grpe vriety used for production of sweet sprkling wines (6 7.5% vol ethnol) nd grown in Piedmont (northwest Itly). The ese nd rpidity of extrction re importnt qulittive prmeters for Brchetto grpes. These romtic red grpes re sujected to very short fermenttion-mcertion (24 48 hr) ecuse the product otined must not exceed 3.5% vol ethnol efore the second fermenttion ( prise de mousse). Reduced mcertion times, low fermenttion temperture (17 18 C) to preserve romtic components, nd low lcohol concentrtions frequently led to reduced pigment extrction nd wines with unstisfctory color. Knowledge of the extrctility of nthocynin compounds mesured vi erry skin hrdness could permit hrvesting t n optiml stge of ripeness nd suitle opertive choices in the prefermenttion nd mcertion phses.

2 94 Rolle et l. Mterils nd Methods Grpe smples. In 2007 Vitis vinifer vr. Brchetto grpe smples were hrvested twice during ripening (with n intervl of 10 dys) from severl vineyrds locted in the Asti nd Alessndri provinces within the Brchetto d Acqui DOCG. Berries were clirted ccording to density (i.e., totl solule solids). Density ws estimted y f lottion of erries in 10 different slt solutions (from 100 to 190 gl -1 NCl) such tht the difference in totl solule solids of two consecutive tches of erries ws ~17 gl -1 (i.e., 1% vol in potentil lcohol) (Fournnd et l. 2006). Two ripening stges were studied: A (250 ± 8 gl -1 sugr) nd B (184 ± 8 gl -1 sugr). Texture nlysis of erry skin. A puncture test ws crried out on the side of ll erries (~200) present in ech one of the two ripening stges defined y flottion (see ove). Mesurements were mde using Universl Testing Mchine TAxT2i Texture Anlyzer (Stle Micro System, Godlming, Surrey, UK) equipped with HDP/90 pltform, needle proe (P/2N), nd 5 kg lod cell. Tests were performed t 1 mms -1 nd erry skin rek force (F sk ), expressed in Newton (N), ws determined (Letief et l. 2008). All dt cquisition ws mde t 400 Hz, using Texture Expert Exceed softwre, version For ech ripening stge, two groups of erries with different skin hrdness were selected: M, soft ( N) nd D, hrd ( N). Anthocynin extrction in hydrolcoholic solution. Sixty erries (three replictes of 20 erries) elonging to ech of the four groups were used for studying nthocynin extrctility. The groups were high sugr, hrd (AD), high sugr, soft (AM), low sugr, hrd (BD), nd low sugr, soft (BM). The erry skins were removed mnully from the pulp nd dried with sorent pper. They were quickly immersed in 75 ml model solution consisting of ethnol/wter (3:97 v/v) to simulte extrction conditions during industril production nd 100 mgl -1 K 2 S 2 O 5 to limit oxidtion of phenolic compounds throughout extrction (Fournnd et l. 2006) nd 5 gl -1 trtric cid nd djusted to ph 3.20 with 1 N NOH. The kinetics of extrction were monitored t regulr intervls: 10, 20, nd 30 min nd 1, 2, 4, 8, 12, 24, nd 48 hr. Spectrophotometric nd HPLC nlysis. A spectrophotometric method ws used to evlute the totl nthocynin index (TAI) of hydrolcoholic solutions nd erry skins efore extrction (Di Stefno nd Crvero 1991). Fresh erry skin nd, t the end of extrction, the residul solid erry skin were quickly immersed in 75 ml of uffer solution contining 12% v/v ethnol, 600 mgl -1 K 2 S 2 O 5, 50 mgl -1 NN 3, nd 5 gl -1 trtric cid nd titrted to ph 3.20 y dding 1 N NOH. After homogeniztion with n UltrTurrx T25 (IKA Lortechnik, Stufen, Germny), the extrct ws centrifuged (1126 g, 10 min, 20 C). The superntnt ws then used for nlysis. Anlysis of individul nthocynins ws performed fter filtrtion using Sep-Pk C18 crtridge (Wters Corportion, Milford, MA) nd elution with methnol. The chromtogrph system ws P100 pump equipped with n AS3000 utosmpler (Spectr Physics Anlyticl, Sn Jose, CA), 20-mL Reodyne smple loop, LiChro- CART column (25 cm x 0.4 cm i.d.) (Merck, Drmstdt, Germny) pcked with LiChrosphere 100 RP-18 5-µm prticles (Alltech, Deerfield, IL), nd Spectr Focus Diode Arry Detector (Spectr Physics) operting t 520 nm. The following conditions were used: solvent A, 10% v/v formic cid in wter; solvent B, 10% v/v formic cid with 50% v/v methnol in wter. These solvents were pssed through 0.20-µm filter. The solvent f low rte ws 1 ml/min nd column temperture ws 20 C. The solvent progrm used ws 72% A to 55% A over 15 min, to 30% A over 20 min, to 10% A over 10 min, to 1% A over 5 min, to 72% A over 3 min. An equilirium time of 10 min ws used (Rolle nd Guidoni 2007). Dt tretment ws crried out using the ChromQuest chromtogrphy dt system (ThermoQuest, Sn Jose, CA). Identifiction of the free forms of nthocynins in the erry skin extrct ws performed y comprison with externl stndrds (delphinidin-3-o-glucoside chloride, mlvidin-3-o-glucoside chloride, peonidin-3-o-glucoside chloride, petunidin chloride, cynidin chloride; Extrsynthèse, Geny, Frnce); the cylted forms of nthocynins were identified y compring the retention time of ech chromtogrphic pek with ville dt in the literture (Di Stefno et l. 1995). The percentges of individul nthocynins were determined y compring the re of ech individul pek with the totl pek re. Sttisticl nlysis. Sttisticl nlysis ws performed using Sttistic for Windows, relese 7.1 (SttSoft, Tuls, OK). A two-wy fctoril nlysis of vrince ws performed to define the interction etween ripening stge nd skin hrdness. Results nd Discussion The concentrtions nd reltive proportions of vrious nthocynins in Brchetto grpes t two ripening stges were determined (Tle 1). Riper grpes (250 gl -1 sugr; A) hd twice the totl nthocynins of less ripe grpes (184 gl -1 sugr; B), nd individul nthocynin content ws different in the two stges. During ripening, mlvidin-3-glucoside nd peonidin-3-glucoside generlly increse in comprison with other nthocynidin monoglucosides (Roggero et l. 1986, Jordão et l. 1998). The rtio of peonidin/mlvidin ws 0.52 in A nd 0.32 in B. Acetylglucoside nthocyninswere the lest undnt clss. Skin rek force (F sk ) vlues were determined for the two ripening stges (Tle 2). Anlysis of vrince did not show significnt differences etween A nd B. The high vriility of skin rek force, under the sme conditions of solule solids present in the erries, is ttriutle to the different culturl nd environmentl situtions of vineyrds from which grpes were tken (Rolle et l. 2006). This vriility ws prt of the experimentl design nd ws intended to provide high distriution of F sk nd to llow the mking of two groups of well-chrcterized erries. Groups M (soft) nd D

3 Skin Brek Force nd Anthocynin Extrctiility 95 Tle 1 Totl nthocynin content nd reltive proportions of individul nthocynins in Brchetto grpes t two ripening stges (A = 250 gl -1 sugr; B = 184 gl -1 sugr) in Mens of three replictes (verge ± stndrd devition). Totl nthocynin index 598 ± ± 76 *** Totl nthocynins (mg kg -1 grpes) Anthocynin profile (%) A B Signf A B Signf Simple glucosides 574 ± ± 0 *** 96.0 ± ± 0.16 ** Acetyl-glucosides 2 ± 0 1 ± 0 ** 0.3 ± ± 0.01 ns Cinnmoyl-glucosides 22 ± 2 15 ± 1 ** 3.7 ± ± 0.17 ** Delphinidin 45 ± 6 16 ± 2 ** 7.5 ± ± 0.59 ** Cynidin 31 ± 2 10 ± 2 ** 5.2 ± ± 0.65 ** Petunidin 44 ± 4 18 ± 1 ** 7.3 ± ± 0.21 ** Peonidin 164 ± ± 7 ** 27.4 ± ± 2.24 ** Mlvidin 315 ± ± 11 ** 52.6 ± ± 3.69 ** **, ***, nd ns indicte significnce t p 0.01, p 0.001, nd not significnt, respectively. Cinnmoyl-glucosides included oth p-coumroyl nd cffeoyl nthocynin forms. Tle 2 Averge, minimum, mximum vlues, nd reltive stndrd devition of skin rek force (F sk ), expressed in Newton (N), of Brchetto grpes t two ripening stges, s determined y puncture test. Skin rek force (N) Avg Min Mx SD A (250 gl -1 sugr) B (184 gl -1 sugr) (hrd) were composed of erries with lower nd higher F sk, respectively, thn the medium vlue of 0.43 N. When vineyrds re more homogeneous, F sk vlues t hrvest hve lower stndrd devitions nd re chrcteristic of the vriety (Letief et l. 2008). Grpe softening during mturtion is the result of significnt chnges in prietl constituent composition, notly in pulp cells (Riéreu- Gyon et l. 2000). Structurl properties of the cell wlls my determine the mechnicl resistnce of erry skin (Brnvon et l. 2000). This chnge during ripening cn e mesured with compression test nd expressed with rheologicl prmeter such s firmness (Roin et l. 1997, Grotte et l. 2001), ut with this type of test, pulp nd skin dt re ggregte. Lee nd Bourne (1980) evluted the evolution of skin hrdness with penetrtion-puncture test using flt proe (0.9 mm dim). This smll cylinder is plunged into the tissue nd mesures the evolution of stress-strin nd mix of compression (under the plunger) nd shering (Roudot 2006). In the puncture test conducted on Brchetto grpes, needle proe ws used to estimte the skin rek force while minimizing other possile interferences. From verison to ripeness, there is n increse in F sk, with stedy or slight decrese round hrvest nd new increse in overripe erries. This generl pttern vries with cultivr (Letief 2007). Totl free nthocynins were extrcted from the four groups of erries (AD, AM, BD, BM) into model hydrolcoholic solution (Tle 3). The extrction of nthocynins is influenced y ethnol concentrtion in wterlcohol solution (Cnls et l. 2005). The finl TAIs of Brchetto grpe extrcts, lthough similr, were strongly ffected y ripening stge, s the initil nthocynin contents were different. Skin hrdness lrgely determined nthocynin relese. Hrd skins under our experimentl conditions relesed 72.4% (group A) nd 70.3% (group B) of the ville nthocynins compred to 62.7% nd 62.1%, respectively, for soft skins. Therefore, skins elonging to group AD presented greter cpcities for nthocynin relese. Some nthocynin types presented different ehviors during extrction. After 48 hr mcertion, in less ripe B erries, peonidin nd cynidin were most undnt in hydrolcoholic solutions otined from hrd skin (Tle 4). In prticulr, the higher TAI in group BD is ttriutle to the greter presence of peonidin. Less drmtic differences in the nthocynin profile were reveled etween hydrolcoholic solutions of groups AM nd AD. In oth ripening stges, significnt differences in cetyl-glucoside nd cinnmoyl-glucoside ssignle to difference of F sk were not oserved, likely ecuse, in Brchetto grpes, these coloring pigments re only present in smll quntities (5%). Ripening stge, skin hrdness, nd the interction etween these two fctors influences the totl mount nd type of individul nthocynin in the finl product (Tle 5). Therefore, the chemicl composition of Brchetto must-wine fter 48 hr mcertion depends not only on erry ripeness ut lso on ll fctors (vintge, environment, vineyrd mngement, clone) tht cn influence skin hrdness (Rolle et l. 2006, Letief et l. 2008, Río Segde et l. 2008). Conclusions For ech ripening stge nd with mcertion in model hydrolcoholic solution, Brchetto grpes with higher skin rek force (F sk ) produced extrcts with higher totl nthocynin. Significnt interctions etween ripening stge nd skin hrdness were found in the individul nthocynin composition of extrcts. Hrd skins were chrcterized y incresed frgility of the cell wlls,

4 96 Rolle et l. TAI (mg mlvidin-3- glucoside L -1 ) Tle 3 Extrctility in model hydrolcoholic solution of totl nthocynin index (TAI) from four groups of erries: AD, AM, BD, BM (A = 250 gl -1 sugr; B = 184 gl -1 sugr; M = soft skin N; D = hrd skin N) (verge ± stndrd devition) nd results of fctoril nlysis of vrince crried out for different extrction times. 10 min 20 min 30 min 1 hr 2 hr 4 hr 8 hr 24 hr 48 hr AM 46 ± 8 85 ± 3 92 ± ± ± ± ± ± ± 24 AD 62 ± ± ± ± ± ± ± ± ± 32 BM 24 ± 3 45 ± ± ± ± ± ± ± ± 11 BD 36 ± 8 65 ± ± ± ± ± ± ± ± 8 Ripening stge ** ** * *** *** *** *** *** *** Hrdness ns ns ns ** ** ** *** ** ** Ripening stge*hrdness ns ns ns ns ns ns ns ns ns Extrction (%) AM 6.6 ± ± ± ± ± ± ± ± ± 3.5 AD 8.9 ± ± ± ± ± ± ± ± ± 4.6 BM 6.0 ± ± ± ± ± ± ± ± ± 3.3 BD 10.5 ± ± ± ± ± ± ± ± ± 2.4 Ripening stge ns ns ns ** * * ** ns ns Hrdness * ns ns * * ** *** ** ** Ripening stge*hrdness ns ns ns ns ns ns ns ns ns *, **, ***, nd ns indicte significnce t p 0.05, p 0.01, p 0.001, nd not significnt, respectively. Tle 4 Anthocynin content nd profile in the hydrolcoholic solution fter 48 hr mcertion for four groups of erries: AD, AM, BD, BM (A = 250 gl -1 sugr; B = 184 gl -1 sugr; M = soft skin N; D = hrd skin N). AM AD Signf BM BD Signf Anthocynin content (mgl -1 ) Totl nthocynin index 434 ± ± 32 ns 215 ± ± 8 * Simple glucosides 421 ± ± 31 ns 206 ± ± 9 * Acetyl-glucosides 2 ± 0 3 ± 0 ns 1 ± 0 1 ± 0 ns Cinnmoyl-glucosides 11 ± 1 12 ± 1 ns 8 ± 2 8 ± 1 ns Delphinidin 30 ± 2 38 ± 3 ns 10 ± 1 10 ± 1 ns Cynidin 23 ± 0 27 ± 3 ns 5 ± 0 8 ± 0 ** Petunidin 31 ± 1 37 ± 3 ns 12 ± 1 12 ± 1 ns Peonidin 140 ± ± 12 ns 41 ± 1 76 ± 6 *** Mlvidin 210 ± ± 12 * 147 ± ± 2 ns Profile (%) Simple glucosides 97.0 ± ± 0.22 ns 95.7 ± ± 0.43 ns Acetyl-glucosides 0.5 ± ± 0.07 ns 0.4 ± ± 0.06 ns Cinnmoyl-glucosides 2.6 ± ± 0.15 ns 3.9 ± ± 0.38 ns Delphinidin 7.2 ± ± 0.18 ** 4.7 ± ± 0.32 ns Cynidin 5.3 ± ± 0.33 ns 2.3 ± ± 0.16 ** Petunidin 7.3 ± ± 0.15 ns 5.5 ± ± 0.27 ** Peonidin 31.3 ± ± 1.10 * 19.2 ± ± 1.46 *** Mlvidin 48.9 ± ± 1.58 ns 68.3 ± ± 1.31 *** *, **, ***, nd ns indicte significnce t p 0.05, p 0.01, p 0.001, nd not significnt, respectively. Cinnmoyl-glucosides include oth p-coumroyl nd cffeoyl nthocynin forms. Tle 5 Fctoril nlysis of vrince crried out on different hydrolcoholic solutions fter 48 hr mcertion. Effect TAI glucosides Simple Acetylglucosides Cinnmoylglucosides Delphinidin Cynidin Petunidin Peonidin Mlvidin Profile (mgl -1 ) Ripening stge *** *** *** ** *** *** *** *** *** Hrdness ** ** ns ns * * * ** * Ripening stge*hrdness ns ns ns ns * ns * ** ** Extrction (%) Ripening stge * ** ** *** *** *** *** *** Hrdness ns ns ns ns ** ns *** *** Ripening stge*hrdness ns ns ns ** ** ** *** *** TAI: totl nthocynin index. *, **, ***, nd ns indicte significnce t p 0.05, p 0.01, p 0.001, nd not significnt, respectively.

5 Skin Brek Force nd Anthocynin Extrctiility 97 which llows greter relese of pigments. Nevertheless, there re no specific studies on the reltionship etween F sk nd the different components of skin cell wlls. Given the ese nd lower cost of the puncture test, skin rek force my e n interesting field prmeter to provide more scientific choice of hrvesting dte. Literture Cited Al, P., J.C. Boulet, nd M. Moutounet Utilistion de prmètres physiques pour l crctéristion de l vérison des ies de risin. J. Int. Sci. Vigne Vin 26: Amrni Joutei, K., nd Y. Glories Tnins et nthocynes: loclistion dns le ie de risin et mode d extrction. Rev. Fr. Œnol. 153: Brnvon, L., T. Doco, N. Terrier, A. Ageorges, C. Romieu, nd P. Pellerin Anlysis of cell wll neutrl sugr composition, β-glctosidse ctivity nd relted cdna clone throughout the development of Vitis vinifer grpe erries. Plnt Physiol. Biochem. 38: Cgnsso, E., L. Rolle, A. Cudn, nd V. Geri Reltions etween grpe phenolic mturity nd red wine phenolic composition. Itl. J. Food Sci. 20: Cnls, S., M.C. Lludy, J. Vlls, J.M. Cnls, nd F. Zmor 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: Di Stefno, R., nd M.C. Crvero Metodi per lo studio dei polifenoli dell uv. Riv. Vitic. Enol. 44: Di Stefno, R., D. Bors, G. Mggiorotto, nd L. Corino Terpeni e polifenoli di uve romtiche frutto colorto prodotte in Piemonte. Enotecnico 29: Fernández-López, J.A., V. Hidlgo, L. Almel, nd J.M. López Roc Quntittive chnges in nthocynin pigments of Vitis vinifer cv Monstrel during mturtion. J. Sci. Food Agric. 58: Fournnd, D., A. Vicens, L. Sidhoum, J.M. Souquet, M. Moutounet, nd V. Cheynier Accumultion nd extrctility of grpe skin tnnins nd nthocynins t different dvnced physiologicl stges. J. Agric. Food Chem. 54: González-Neves, G., D. Chrmelo, J. Bldo, L. Brreiro, R. Bochicchio, G. Gto, G. Gil, A. Tessore, A. Croneu, nd M. Moutounet Phenolic potentil of Tnnt, Cernet Suvignon nd Merlot grpes nd their correspondence with wine composition. Anl. Chim. Act 513: Grotte, M., Y. Cdot, A. Poussier, D. Loonis, E. Piétri, F. Duprt, nd G. Breu Détermintion du degré de mturité des ies de risin pr des mesures physiques: spects méthodologiques. J. Int. Sci. Vigne Vin 35: Guidoni, S., A. Ferrndino, nd V. Novello Effects of sesonl nd gronomicl prctices on skin nthocynin profile of Neiolo grpes. Am. J. Enol. Vitic. 59: Jordão, A.M., J.M. Ricrdo D Silv, nd O. Lureno Evolution of nthocynins during grpe mturtion of two vrieties (Vitis vinifer L.), Cstelo Frnces nd Tourig Frnces. Vitis 37: Le Moigne, M., C. Mury, D. Bertrnd, nd F. Jourjon Sensory nd instrumentl chrcteristion of Cernet frnc grpes ccording to ripening stges nd growing loction. Food Qul. Pref. 19: Lee, C.Y., nd M.C. Bourne Chnges in grpe firmness during mturtion. J. Text. Stud. 11: Letief, H Appliction of chemicl-physicl nd mechnicl tests for the definition of wine grpe qulity. PhD thesis, University of Turin. Letief, H., L. Rolle, G. Zepp, nd V. Geri Assessment of the grpe skin hrdness y puncture test. J. Sci. Food Agric. 88: Letief, H., L. Rolle, nd V. Geri Mechnicl ehvior of winegrpes under compression tests. Am. J. Enol. Vitic. 59: Orteg-Regules, A., I. Romero-Cscles, J.M. Ros-Grcí, J.M. López-Roc, nd E. Gómez-Plz A first pproch towrds the reltionship etween grpe skin cell-wll composition nd nthocynin extrctility. Anl. Chim. Act 563: Orteg-Regules, A., J.M. Ros Grcí, A.B. Butist-Ortin, J.M. López-Roc, nd E. Gómez-Plz Chnges in skin cell wll composition during the mturtion of four premium wine grpe vrieties. J. Sci. Food Agric. 88: Riéreu-Gyon, P., Y. Glories, A. Mujen, nd D. Duourdieu Hndook of Enology. Vol. 2. The Chemistry of Wine Stiliztion nd Tretments, pp Wiley & Sons, Chichester, UK. Río Segde, S., L. Rolle, V. Geri, nd I. Orriols Phenolic ripeness ssessment of grpe skin y texture nlysis. J. Food Comp. Anl. 21: Roin, J.P., P. Al, nd J.M. Slmon Fermeté et mturtion du risin. Définition et évolution de différents prmètres rhéologiques u cours de l mturtion. J. Int. Sci. Vigne Vin. 31: Roggero, J.P., S. Coen, nd B. Rgonnet High performnce liquid chromtogrphy survey on chnges in pigment content in ripening grpes of Syrh. An pproch to nthocynin metolism. Am. J. Enol. Vitic. 37: Rolle, L., H. Letief, E. Cgnsso, D. Ghirrdello, G. Zepp, nd V. Geri Studio delle proprietà meccniche di uve Neiolo coltivte in mienti diversi. Qud. Vitic. Enol. Univ. Torino. 28: Rolle, L., nd S. Guidoni Color nd nthocynin evlution of red winegrpes y CIE L*, *, * prmeters. J. Int. Sci. Vigne Vin 41: Romero-Cscles, I., J.I. Fernández-Fernández, J.M. López-Roc, nd E. Gómez-Plz The mcertion process during winemking. Extrction of nthocynins from grpe skins into wine. Eur. Food Res. Technol. 221: Romero-Cscles, I., A. Orteg-Regules, J.M. López-Roc, J.I. Fernández-Fernández, nd E. Gómez-Plz Differences in nthocynin extrctility from grpes to wines ccording to vriety. Am. J. Enol. Vitic. 56: Roudot, A.C Some considertions for theory of plnt tissue mechnics. Sci. Aliments 26: Venencie, C., B. Videu, nd D. Michel Contrôle mturité: nlyse des pellicules ou des ies entiéres? Rev. Fr. Oenol. 169:13-15.