The aim of this work was to study the effect of the use of pectolytic enzymes on the phenolic composition of young white wines made of white grape Smederevka (V. vinifera L.). White grape mash from Smederevka were macerated for 4 hours at 18 to 20 oC with addition on one commercial pectolytic enzyme preparation (Vinozym Process, Trenolin Mash DF, Rohavin LX).

The obtained results of laboratory trials showed decreased concentration of total phenolics by 37.8% and results of industry trials decreased by 39.2%. These enzyme treatments resulted in clarified the white grape juice, degraded the grape mash, reduced juice viscosity and total phenols and elicited an improvement in the visual aspect of the wine

Božinović, Zvonimir

Ivanova, Violeta

Mojsov, Kiro

Petreska, Meri

Ziberoski, Jugoslav

UGD Academic Repository

Effects of pectolytic enzyme treatments on white grape mashs of Smederevka on polyphenolic content of wines Section 8 . Viticulture and Enology 963Effects of pectolytic enzyme treatments on white grape mashs of Smederevka on polyphenolic content of wines Kiro MOJSOV1, Jugoslav ZIBEROSKI2, Zvonimir BOZINOVIC2, Violeta IVANOVA3, Meri PETRESKA4 1University “Goce Delcev” Stip, Technological-Technical Faculty, Krste Misirkov bb, 2000 Stip, Republic of Macedonia, (e-mail kiro.mojsov@ugd.edu.mk) 2Ss. Cyril and Methodius University, Faculty of Agricultural Sciences and Food, Aleksandar Makedonski bb, 1000 Skopje, Republic of Macedonia 3Ss. Cyril and Methodius University, Institute of Agriculture, Aleksandar Makedonski bb, 1000 Skopje, Republic of Macedonia 4Ministry of Agriculture, Forestry and Water Economy, Leniniva 2, 1000 Skoje, R. Macedonia Abstract The aim of this work was to study the effect of the use of pectolytic enzymes on the phenolic composition of young white wines made of white grape Smederevka (V. vinifera L.). White grape mash from Smederevka were macerated for 4 hours at 18 to 20 oC with addition on one commercial pectolytic enzyme preparation (Vinozym Process, Trenolin Mash DF, Rohavin LX). The obtained results of laboratory trials showed decreased concentration of total phenolics by 37.8% and results of industry trials decreased by 39.2%. These enzyme treatments resulted in clarified the white grape juice, degraded the grape mash, reduced juice viscosity and total phenols and elicited an improvement in the visual aspect of the wine. Key words: pectolytic enzymes, wine-making, total phenolics, flavonoids, catechins Utjecaj pektolitičkih enzima u masulju grožđa Smederevka na koncentraciju polifenola u vinu Sažetak Cilj ovog rada bio je odrediti utjecaj pektolitičkih enzima na fenolni sastav mladih vina Smederevka (V. vinifera). Masulj grožđa Smederevka maceriran je 4 sata na 18 do 20 oC sa dodatkom jednog komercijalnog pektolitičkog enzimskog preparata (Vinozym Process, Trenolin Mash DF and Rohavin LX). Dobiveni rezultati laboratorijskog pokusa pokazali su smanjenje koncentracije ukupnih fenola od 37.8%, a rezultati industrijskog pokusa smanjenje od 39.2%. Primjena pektolitičkih enzima utjecala je na bistroću mošta, smanjenje viskoziteta i ukupnih fenola te su pozitivno utjecali na vizualni izgled vina. Ključne riječi: pektolitički enzimi, vino, polifenoli, Smederevka     Proceedings. 46th Croatian and 6th International Symposium on Agriculture. Opatija. Croatia (963-967) ORIGINAL SCIENTIFIC PAPERKiro MOJSOV, Jugoslav ZIBEROSKI, Zvonimir BOZINOVIC, Violeta IVANOVA, Meri PETRESKA 46th Croatian and 6th International Symposium on Agriculture 964 Introduction Smederevka is a variety of white grape cultivated in Republic of Macedonia used for production of quality wines. Although the composition of the grape depends on its variety, the soil and climatic conditions, there is little variation in the actual cell structure of the plant. Wines and grapes contain a number of polyphenolic constituents classified as flavonoids (anthocyanins, flavan-3-ol-monomers and polymers, flavonols and dihydroflavonols) and non-flavonoids (hydroxybenzoic acid and hydroxycinnamic acid and their derivatives, stilbenes and phenolic alcohols) that play a mayor role in enology. Red wines contain all the above phenolics, while white wines contain mainly phenolic acids and flavanols (Ribéreau-Gayon et al., 2000). Wine phenolics are important quality components that contribute to the colour, taste, and feel of wines. Pectolytic enzyme preparations are the most widely used in fruit and wine processing industry and in general are the most efficient at degrading polysaccharide. Numerous studies have reported on the use of commercial enzymes in white grape juice clarification, enzyme treatment effects on quality of white grape musts and wines, effects of different commercial pectolytic enzyme preparations on white grape musts and wines et al. (Ough C. and Crowell E., 1979; Brown M. and Ough C., 1981; Sreenath H. and Krishnaswamy S., 1992; Lao et al., 1997). Treating crushed white grapes with pectic enzyme increases juice yield, sensory tests indicate that wine quality is either enhanced or unchanged (Ough C. and Crowell E., 1979). White grape musts of eight different varieties were treated with pectic enzyme preparations.These treatments resulted in increases in total juice yields, clarify of the wine, filterability, methanol production, wine quality, browning capacity and amounts of solids that settled (Brown M. and Ough C., 1981). A commercial pectinase from Aspergillus niger containing various polysaccharases clarified the white grape juice to an extent of 98-99% and also degraded the grape mash by 25-30%. After pectinolytic juice clarification, both juice viscosity and total phenols were reduced by 25% and 32% respectively (Sreenath H. and Krishnaswamy S., 1992). Grapes of three white cultivars were treated with a commercial pectic enzyme preparation on an industrial scale. Pectic enzyme treatment increased the higher alcohols, free hydroxycinnamic acids and volatile phenols, whereas amounts of most esters, herbaceous alcohols and hydroxycinnamate derivative esters were reduced. When tasted by an expert panel, differences were found in sensory characteristics, with wines from untreated judged higher quality than those from treated grapes (Lao et al., 1997). Enzyme treatments on mushs speed up settling and ensure efficient sediment compaction (Cruess et al., 1951; Höhn et al., 2005; Trepo, 2008). The clear must is then racked from the lees without problems. Material and methods Commercial pectolytic enzyme preparations In this study were used for laboratory trials three commercial macerating pectolytic enzyme preparations with four doses: Vinozym Process, Novozymes A/S, Bagsvaerd, Denmark; Doses: 3, 4, 5, and 6 g/100 kg grapes (I-1, I-2, I-3, I-4) Trenolin Mash DF, Erbslöh Geisenheim AG, Geisenheim, Germany); Doses: 1, 2, 3, and 4 mL/100 kg grapes (II-1, II-2, II-3, II-4) Rohavin LX, AB Enzymes GmbH, Darmstadt, Germany; Doses: 2, 3, 4, and 5 mL/100 kg grapes (III-1, III-2, III-3, III-4) and for industrial trials used one commercial pectolytic enzyme preparation (Trenolin Mash DF, 2 mL/100 kg grapes) along with controls with no added enzyme(IV). These enzyme preparations are derived from cultures of Aspergillus niger which is a species accepted as G.R.A.S. (Generally Recognized As Safe) (Canal-Llauberes, 1993). Grape samples for laboratory trials The white grape cultivar Smederevka (Vitis vinifera), cultivated in the Ovce pole vineyard, the Povardarie region, Republic of Macedonia, were harvested at optimal maturity (2009 vintage), at 170-190 g L-1 sugar, 6.0-7.0 g L-1 total acids, and pH from 3.0 to 3.2, and transported to the private winery “Imako Vino” Stip, Republic of Macedonia. Effects of pectolytic enzyme treatments on white grape mashs of Smederevka on polyphenolic content of wines Section 8 . Viticulture and Enology 965Grape samples for industrial trials The white grape cultivar Smederevka (Vitis vinifera), cultivated in the Veles vineyard, the Povardarie region, Republic of Macedonia, were harvested at optimal maturity (2009 vintage), at 180-200 g L-1 sugar, 6.0-7.0 g L-1 total acids, and pH from 3.0 to 3.2, and transported to the private winery “Tristo” Veles, Republic of Macedonia. Vinification The grapes were weighed, destemmed, crushed and divided in 5 liters plastic reservoirs for laboratory trials, and for industrial trials were placed in a stainless steel fermentor (4 t.). All laboratory treatments were performed in triplicate and industrial in duplicate. White grape mash made from Smederevka were macerated for 4 hours at 18 to 20 oC with addition on one commercial pectolytic enzyme preparation. Control laboratory trials were in all same with experimental trials only whitout added pectolytic enzyme preparation. After maceration the pomace was removed and each must are pour into the funnel and collect musts in 5 liters plastic reservoirs (3 for enzyme treatments musts and 1 for control must (no-treatment). In each reservoir are add 30 ppm SO2 and are keept musts cool (15-16 oC) and allow to stand overnight, so that suspended material will fall to the bottom. The clear must is then racked from the lees without problems. In each musts (reservoir) are add yeast (Saccharomyces cerevisiae) NEUTRE SC (Lallemand) (200 mg/kg grapes) at ~25 oC to completion of fermentation. The bottled wines (0.5 l) were stored at 4 - 6 oC, and phenolic compounds were measurmed after 6 months wine maturation. Instrumentation and reagents Analysis of polyphenolic components was carried out with a Aglient 8453 UV-Vis spectrophotometer. All analyses were performed in duplicate. The reagent p-(dimethylamino)cinnamaldehyde (p-DMACA), standards of gallic acid and (+)-catechin were from Fluka, and the Folin-Ciocalteu reagent was from Merck. All other used reagents were of analytical purity grade. Total phenolics assay The Folin-Ciocalteu method was used for the determination of the total phenolics (Ivanova et al., 2010). Total flavonoids assay Total flavonoids were determined using the colorimetric assay with aluminium chloride and (+)-catechin as standard for calibration according to Zhishen et al., (1999). Total catechins assay The concentration of total catechins in wines was determined using the method of Di Stefano et al. (1989) with the reagent p-DMACA. Results and discussion Spectrophotometric determinations of total phenolics (TP), total flavonoids (TF), total catechins (TC), were performed for the Smederevka wines obtained with pre-fermentation enzyme maceration (4 hours) on grape mashs, and after alcoholic fermentation to completion of fermentation. Measurements were performed in order to study the effects of three different macerating enzymes on polyphenol contents of Smederevka wines from Macedonia compared to control wines no added enzyme. The obtained results of laboratory trials (Table 1) showed decreased concentration of total phenolics (TP) from 699.1 mg L-1 to 422.5 mg L-1 (37.8%) compared to the control samples from 679.1 mg L-1. Pectolytic enzyme preparation Vinozym Process (from 699.1 mg L-1 to 558.8 mg L-1, decreased 17.7%) Trenolin Mash DF (from 550.2 mg L-1 to 488.1 mg L-1, decreased 28.1%) and Rohavin LX (from 483.7 mg L-1 to 422.5 mg L-1, decreased 37.7%), depend of used doses. Kiro MOJSOV, Jugoslav ZIBEROSKI, Zvonimir BOZINOVIC, Violeta IVANOVA, Meri PETRESKA 46th Croatian and 6th International Symposium on Agriculture 966 Table 1. Effects of pectolytic enzyme treatments on white grape mashs of Smederevka on phenolic content of wines (TP: total phenolics, TF: total flavonoids, TC: total catechins). Laboratory trials: 5 kg grape Enzyme preparations Dose  аTP mg L-1 аTF mg L-1 аTC mg L-1 Vinozym Process I-1 558.8 ± 0.25 54.83 ± 0.02 5.70 ± 0.06 3;4;5;6 g/100kg grapes I-2 699.1 ± 1.35 91.46 ± 0.01 8.49 ± 0.15  I-3 595.2 ± 0.00 83.06 ± 0.17 7.39 ± 0.00  I-4 604.0 ± 0.45 64.91 ± 0.03 6.37 ± 0.28 Trenolin Mash DF II-1 550.2 ± 0.50 66.17 ± 0.08 6.99 ± 0.03 1;2;3;4 ml/100kg grapes II-2 488.1 ± 0.95 39.86 ± 0.02 4.26 ± 0.02  II-3 521.9 ± 1.40 40.30 ± 0.02 4.06 ± 0.03  II-4 512.5 ± 1.75 62.05 ± 0.03 7.21 ± 0.02 Rohavin LX III-1 449.6 ± 0.35 38.63 ± 0.03 3.65 ± 0.01 2;3;4;5 ml/100kg grapes III-2 422.5 ± 1.30 33.40 ± 0.02 3.48 ± 0.07  III-3 483.7 ± 0.55 38.43 ± 0.12 4.49 ± 0.01  III-4 453.5 ± 1.25 33.33 ± 0.02 3.84 ± 0.00 Control-no added enzyme IV 679.1 ± 2.55 80.12 ± 0.99 8.74 ± 0.03 а the values are average from 2 replicates±SD Table 2. Effects of pectolytic enzyme treatments on white grape mashs of Smederevka on phenolic content of wines (TP: total phenolics, TF: total flavonoids, TC: total catechins). Industrial trials: 1220 kg grape. Private winery “Tristo” Veles Enzyme preparations  Dose  аTP mg L-1 аTF mg L-1 аTC mg L-1 Trenolin Mash DF 2 ml/100kg grapes II-2 341.6 ± 2.65 40.81 ± 0.12 4.09 ± 0.03 Control-no added enzyme IV 562.3 ± 2.50 87.63 ± 0.16 11.55 ± 0.07 а the values are average from 2 replicates±SD The obtained results of industry trials(Table 2) showed decreased concentration of total phenolic (TP) from 341.6 mg L-1 compared to the control sample from 562.3 mg L-1 (decreased 39.2%). These enzyme treatments resulted in clarified the white grape juice, degraded the grape mash, reduced juice viscosity and total phenols and elicited an improvement in the visual aspect of the wine. The obtained results for the analysed wines were in agreement with previously published data (Ough C. and Crowell E., 1979; Brown M. and Ough C., 1981; Sreenath H. and Krishnaswamy S., 1992; Lao et al., 1997). Conclusion The application of pectolytic enzymes would facilitate break up of the grape cell wall and aid juice and wine clarification by breaking-down the released grape pectins. Enzyme treatment also resulted in white wines with reduced juice viscosity and total phenols and elicited an improvement in the visual aspect of the wine. Acknowledgement We are acknowledge on a Private Winery. Many thanks to Risto Ljubotenski, owner on Prvate Winery “Imako vino” Stip (Macedonia), for support, providing the grapes and permit for conducting on laboratory trials in his winery.. Also many thanks for producers on enzyme preparations Novozymes (Denmark), Erbslöh (Germany), and AB Enzyme (Germany) for delivered enzyme preparations. References Brown M.R., Ough C.S. 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Effecs of pectolytic enzyme treatments on white grape mashes of Smederevka on polyphenolic content of wines

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