Two strains of Saccharomyces cerevisiae were employed for winemaking of must from red grapes. Twenty-two parameters were determined in the red wines produced. Very significant (p<0.01) differences were observed for colour intensity, total polyphenols, and non-anthocyanic flavonoids. Moreover, significant (p<0.05) differences were observed for colour and monomeric anthocyanins

Andrea Caridi

Antonio Cufari

Idolo Tedesco

Raffaele Lovino

Rosanna Palumbo

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Influence of Yeast on Polyphenol Composition of Wine

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UDC 663.541.2:547.56:663.222 preliminary communicationISSN 1330-9862(FTB-1273)Influence of Yeast on Polyphenol Composition of Wine**Andrea Caridi1*, Antonio Cufari1, Raffaele Lovino2,Rosanna Palumbo3,4 and Idolo Tedesco41Dipartimento di Scienze e Tecnologie Agro-Forestali e Ambientali (STAFA), Università Mediterranea diReggio Calabria, Piazza San Francesco 7, I-89061 Gallina (RC), Italia2Istituto Sperimentale per l’Enologia, Sezione Operativa di Barletta,Via Vittorio Veneto 26, I-70051 Barletta (BA), Italia3Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 6, I-80136 Napoli (NA), Italia4Istituto di Scienze dell’Alimentazione, Consiglio Nazionale delle Ricerche,Via Roma 52A/C, I-83100 Avellino (AV), ItaliaReceived: October 2, 2003Accepted: February 16, 2004SummaryTwo strains of Saccharomyces cerevisiae were employed for winemaking of must fromred grapes. Twenty-two parameters were determined in the red wines produced. Very sig-nificant (p<0.01) differences were observed for colour intensity, total polyphenols, andnon-anthocyanic flavonoids. Moreover, significant (p<0.05) differences were observed forcolour and monomeric anthocyanins.Key words: fermentation, phenolics, red wine, wine yeast37A. CARIDI et al.: Influence of Yeast on Polyphenols in Wine, Food Technol. Biotechnol. 42 (1) 37–40 (2004)* Corresponding author; Phone: ++39 0965 682 816; Fax: ++39 0965 680 727; E-mail: acaridi@unirc.it** This paper was presented at the 1st FEMS Congress in Ljubljana, Slovenia, June 29 – July 3, 2003IntroductionIn the production of red wine, the type and quantityof phenolics play a major role in the quality of wine.Anthocyanins, flavonols, catechins and other flavonoidscontribute to the sensory characteristics of wine, particu-larly colour and astringency; in addition, they possess awide range of antioxidant and pharmacological effects.Phenolics vary notably according to several parame-ters (1,2) such as the grape variety (3), the macerationtemperature (4), the length of grape pomace contact (5)and other vinification conditions (6–8). During ageing,phenolics evolve and monomeric anthocyanins polymer-ize by reaction with other flavonoid compounds and al-dehydes (9).It is well known that wine yeasts are among thecauses that decrease the phenolic content of wines (10).This mechanism can be exclusively physical, involvingthe establishment of weak and reversible interactionsmainly between anthocyanins and yeast walls by ab-sorption (11). Various yeast metabolites, such as pyruvicacid (12) and acetaldehyde (13,14), were shown to reactwith different classes of phenolics, suggesting that theyoffer an important way of stabilizing pigments duringthe maturation and ageing of wine. Furthermore, an en-zymatic hydrolysis involving a yeast periplasmic antho-cyanin-b-D-glucosidase, followed by a decolourizing ac-tivity connected to the loss of A520nm, was described (15).Heterologous expression of an anthocyanin-b-D-glucosi-dase in a wine yeast strain was performed, but withoutobtaining wines with different physicochemical charac-teristics (16). The effect of four strains of Saccharomycescerevisiae on phenolic glycosides was evaluated duringfermentation and during ageing of yeast for 40 months,but no differences among strains were observed (17).The influence of yeast used for winemaking on pheno-lics is still poorly understood; so we decided to verify ifdifferent wine yeasts can somewhat modify chromaticproperties, phenolic profile and antioxidant power ofwine.In this paper, we have analyzed the wines producedusing two different wine yeasts and Gaglioppo mustfrom red grapes. This variety was employed because ithas limited content of anthocyanins; so Gaglioppo winesare very susceptible to browning and, when elaboratedfor young wines, they lose their vivid colour rapidly.Material and MethodsMicroorganismsThe work was performed using two different strainsof Saccharomyces cerevisiae, Sc2659 and Sc1483; thesewine yeasts belong to the Department STAFA collection(Reggio Calabria, Italia).Winemaking testAbout 1600 kg of Gaglioppo grapes (Vitis viniferaL.), cultivated in Cirò Marina (South of Italy), were har-vested at optimum maturity and transported to the win-ery. Grapes were processed using a mechanical cru-sher/destemmer. Crushed grapes were subdivided intofour 400-kg lots and dispensed into stainless steel ves-sels of 600 L to provide two replicates of the two wineyeasts used. Then, the must was treated with SO2 (50mg/kg) and inoculated with 5 % of 48-h yeast precultu-res. The contact with grape pomace was prolonged forthe complete fermentation period of 18 days; fermenta-tion caps were punched down six times the first day,five times the second day, four times the third day,three times the fourth day, twice the fifth, sixth and sev-enth days, and once the following days. Fermentationswere monitored by determining the increase of tempera-ture and the decrease of °Brix. At the end of fermenta-tion the grape pomace was pressed and, after spontane-ous sedimentation, the wines were racked, bottled andimmediately analyzed.Wine analysisOn the whole, 22 parameters were determined inthe wines obtained with the two yeasts. Acetic acid,succinic acid and glycerol were analyzed using specificBoehringer kits on diluted samples. The chromatic prop-erties of the wines were determined according to theEEC methods (18) and expressed as follows: the colourwas given by the value of A520nm, the colour intensitywas given by the sum of the A420nm, A520nm, and A620nm,the tint was expressed by the ratio of the A420nm andA520nm. The total polyphenol content was determined us-ing the Folin-Ciocalteu reagent according to Singletonand Rossi (19). The non-flavonoid compounds were de-termined according to Kramling and Singleton (20). Theflavonoid compound content was calculated by differ-ence between the total polyphenols and the non-flavo-noid compounds. Values were expressed as concentra-tion (M) of gallic acid equivalent. In order to determinein vitro antioxidant properties of red wines, a colouri-metric method based on the reduction of a ferrictripyridyl-s-triazine complex to its ferrous form, namelyFRAP (ferric reducing antioxidant power) assay (21),was employed. A volume of 10 L of wine (diluted inratio 1:10 with ethanol 10 %) was added to the volumeof 1 mL of FRAP assay solution, prepared by mixing 25mL of 300 mM acetate buffer (pH=3.6), 2.5 mL of2,4,6-tripyridyl-s-triazine (TPTZ) solution (10 mM TPTZin 40 mM HCl) and 2.5 mL of 20 mM FeCl3·6H2O. Anti-oxidant power was calculated as difference in the A593nmafter 0 and 6 min. The values were correlated to a stan-dard curve made with a pure solution of quercetin. Allthe other parameters were determined using standardmethods (22,23). Analyses were performed in triplicatefor each sample and data were subjected to mathemati-cal and statistical analysis.Results and DiscussionThe grape must had the following characteristics:23.6 °Brix, 7.31 g/L titratable acidity, pH=3.63. Duringthe 18 days of winemaking the temperature oscillatedfrom a minimum of 20 °C to a maximum of 24 °C, withsimilar profiles for all the four vessels. The decrease ofthe °Brix was somewhat faster for the two vessels inocu-lated with strain Sc2659, according to its higher fermen-tation rate. The physicochemical parameters of the redwines produced with the two wine yeasts are reportedin Table 1; strain Sc2659 would be preferable comparedto strain Sc1483 because it produced significantly higherlevels of ethanol (p<0.01) and lower levels of acetic acid(p<0.05). Significantly higher (p<0.01) differences in to-tal dry extract clearly depend on the different content inresidual reducing sugars (p<0.01); effectively, a compar-ison between the sugar-free extracts, i.e. the differencebetween the total dry extract and the total sugars, didnot show significant differences.The chromatic properties, the phenolic profile, andthe antioxidant power of the red wines produced withthe two wine yeasts are reported in Table 2. StrainSc2659, compared to strain Sc1483, produced a winewith significantly higher values of colour, colour inten-sity, total polyphenols and monomeric anthocyanins.Also, the content of flavonoids, total anthocyanins,flavans and proanthocyanidins was higher in the wineproduced by strain Sc2659, but the differences from thestrain Sc1483 were not significant. The levels of non--anthocyanic flavonoids were significantly lower. There-fore, strain Sc2659 protects, during winemaking, thephenolics and, above all, the anthocyanins of the mustbetter than strain Sc1483. Obviously, colour and colourintensity are also notably influenced by this behaviour,because anthocyanins are the major components respon-sible for red wine colour (24). In the wine producedwith strain Sc2659 the monomeric anthocyanins andflavans are higher: this probably indicates low absorp-tion of these classes of phenolics on the yeast walls,which probably delayed the polymerization process.This effect could be very useful in winemaking of grapemust with low levels of anthocyanins and high percent-ages of a few stable di-substituted anthocyanins, such asthe Gaglioppo variety (25). Low level of non-anthocyanicflavonoids and, at the same time, high content of38 A. CARIDI et al.: Influence of Yeast on Polyphenols in Wine, Food Technol. Biotechnol. 42 (1) 37–40 (2004)proanthocyanidins could produce a more astringentwine. Interestingly, the strain 1483, which fermented ata slower rate, gave lower phenolic protection; Mazza etal. (24) found a similar behaviour for strain Wädenswil27 used to ferment Pinot noir must, however, they con-cluded that yeast used for fermentation had minimal ef-fect.The antioxidant properties of wines are usually in-vestigated according to Rice-Evans and Miller (26); ne-vertheless, there are many methods to determine antiox-idant capacity (27). Methods differ in terms of the assayprinciples and experimental conditions; consequently, indifferent methods specific antioxidants have varyingcontributions to total antioxidant potential (28). Previousworks reported that FRAP assay is a valid method todetermine the antioxidant properties of red wine (29,30).Therefore, we used this method to measure the antioxi-dant activity of red wines obtained with the two differ-ent strains. As indicated in Table 2, wine obtained withstrain Sc2659 showed the highest FRAP value; the anti-oxidant effects appear to depend on the concentration offlavonoids but not on simple phenolic compounds, ac-cording to a previous work (31).These preliminary results showed interesting corre-lations between yeast strain used for winemaking andphenolic composition of wine, elucidating that strain be-haviour can somewhat modify chromatic properties,phenolic profile and antioxidant power of wine.AcknowledgementsThe authors thank the wineries Vincenzo Ippolito(KR) for their kind technical assistance during winemak-ing. This work was supported by a grant from the Euro-pean Community, European Agricultural Guidance andGuarantee Fund, and from the Italian Government,Ministero per le Politiche Agricole, for the Research Pro-ject POM B35: »Miglioramento e valorizzazione dei viniottenuti da uve autoctone dell’Italia meridionale attra-verso lo studio ed il controllo delle variabili critiche chene determinano la tipicità sensoriale«.References1. V. Cheynier, I. H. Arellano, J. M. Souquet, M. Moutounet,Am. J. Enol. Vitic. 48 (1997) 225–228.2. J. M. Auw, V. Blanco, S. F. O'Keefe, C. A. Sims, Am. J.Enol. Vitic. 47 (1996) 279–286.39A. CARIDI et al.: Influence of Yeast on Polyphenols in Wine, Food Technol. Biotechnol. 42 (1) 37–40 (2004)Table 1. Physicochemical parameters in red wines obtained with two different wine yeastsParameters Sc2659 Sc1483Mean SD Mean SDpH 3.74 0.01 3.69 0.03j(ethanol)/% 14.20 A 0.14 12.89 B 0.09g(reducing sugars)/(g/L) 8.5 B 0.21 23.0 A 0.85g(total dry extract)/(g/L) 45.0 B 0.07 59.7 A 1.13g(sugar-free extract)/(g/L) 37.5 0.14 37.7 0.28g(titratable acidity)/(g/L) 6.51 0.01 6.60 0.04g(total SO2)/(mg/L) 15 1.41 11 1.41g(acetic acid)/(g/L) 0.37 b 0.04 0.59 a 0.01g(succinic acid)/(g/L) 0.44 0.12 0.64 0.15g(glycerol)/(g/L) 7.05 0.33 7.50 0.08A, B: p<0.01; a, b: p<0.05 indicate the significance of the comparison in the same rowTable 2. Chromatic properties, phenolic profile, and antioxidant power in red wines obtained withtwo different wine yeastsParameters Sc2659 Sc1483Mean SD Mean SDColour 2.49 a 0.05 1.95 b 0.08Tint 1.03 0.06 1.14 0.09Colour intensity 5.76 A 0.08 4.67 B 0.10(total polyphenols)/(mg/L) 3900 A 96.87 3156 B 28.28c(flavonoids equiv. to gallic acid)/M 22.40 0.71 16.30 2.26c(non-flavonoids equiv. to gallic acid)/M 0.57 0.01 0.60 0.01g(non-anthocyanic flavonoids)/(mg/L) 1419 B 7.78 2059 A 87.68g(total anthocyanins)/(mg/L) 80.2 2.40 74.0 1.13g(monomeric anthocyanins)/(mg/L) 23.4 a 1.48 16.3 b 0.28g(flavans)/(mg/L) 2625 219.20 2171 34.65g(proanthocyanidins)/(mg/L) 4367 236.17 3816 357.09c(FRAP value equiv. to quercetin)/M 6.00 0.42 4.13 0.67A, B: p<0.01; a, b: p<0.05 indicate the significance of the comparison in the same row3. G. Mazza, Crit. Rev. Food Sci. Nutr. 35 (1995) 341–371.4. C. S. Du Plessis, Die Wynboer, 449 (1973) 11–13.5. P. D. Scudamore-Smith, R. L. Hooper, E. D. McLaran, Am.J. Enol. 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Tech. 35 (2000) 95–103.16. P. Sanchez-Torres, L. Gonzalez-Candelas, D. Ramon, J.Agric. Food Chem. 46 (1998) 354–360.17. B. W. Zoecklein, C. H. Hackney, S. E. Duncan, J. E. Marcy,J. Ind. Microbiol. Biotechnol. 22 (1999) 100–107.18. EEC, Commission Regulation N° 2676/90 of 17 September1990 determining Community methods for the analysis of wi-nes, Official Journal L 272, 03/10/1990, 1–192.19. S. L. Singleton, J. A. Rossi, Am. J. Enol. Vitic. 16 (1965)144–158.20. T. E. Kramling, V. L. Singleton, Am. J. Enol. Vitic. 20 (1969)86–92.21. I. F. F. Benzie, J. J. Strain, Anal. Biochem. 239 (1996) 70–76.22. C. S. Ough, M. A. Amerine: Methods for Analysis of Mustsand Wines, John Wiley and Sons, New York (1988).23. R. Di Stefano, M. C. Cravero, N. Gentilini, L'Enotecnico, 25(1989) 83–89.24. G. Mazza, L. Fukumoto, P. Delaquis, B. Girard, B. Ewert,J. Agric. Food Chem. 47 (1999) 4009–4017.25. R. Lovino, E. La Notte, S. Suriano, M. Savino, P. Dimitri,Proceedings of the 2nd Workshop on »Miglioramento e valoriz-zazione dei vini ottenuti da uve autoctone dell'Italia meridionaleattraverso lo studio ed il controllo delle variabili critiche che nedeterminano la tipicità sensoriale«, Foggia, Italia (2001) pp.36–54.26. C. Rice-Evans, N. J. Miller, Methods Enzymol. 234 (1994)279–293.27. H. Zielifski, H. Kozowska, Pol. J. Food Nutr. Sci. 8 (1999)147–158.28. G. Cao, R. L. Prior, Clin. Chem. 44 (1998) 1309–1315.29. A. Arnous, D. P. Makris, P. Kefalas, J. Agric. Food Chem. 49(2001) 5736–5742.30. I. Tedesco, G. L. Russo, F. Nazzaro, M. Russo, R. Palumbo,J. Nutr. Biochem. 12 (2001) 505–511.31. I. Tedesco, M. Russo, P. Russo, G. Iacomino, G. L. Russo,A. Carraturo, C. Faruolo, L. Moio, R. Palumbo, J. Nutr.Biochem. 11 (2000) 114–119.Utjecaj sojeva kvasca na sastav polifenola u vinuSa`etakZa proizvodnju vina iz mo{ta dobivenog od crvenoga gro`|a upotrijebljena su dvasoja Saccharomyces cerevisiae. U proizvedenim crvenim vinima odre|ena su 22 fizikalno-ke-mijska i polifenolna parametra. Od polifenolnih parametara vrlo zna~ajne razlike (p<0,01)opa`ene su u intenzitetu boje, ukupnim polifenolima i neantocijanskim flavonoidima. Na-dalje, zna~ajne razlike (p<0,05) uo~ene su za boju i monomerne antocijanine.40 A. CARIDI et al.: Influence of Yeast on Polyphenols in Wine, Food Technol. Biotechnol. 42 (1) 37–40 (2004)

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