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. 2019 Feb 20;56(4):1841–1853. doi: 10.1007/s13197-019-03638-4

Effect of winemaking on phenolic profile, colour components and antioxidants in Crljenak kaštelanski (sin. Zinfandel, Primitivo, Tribidrag) wine

Ivana Generalić Mekinić 1,✉, Živko Skračić 2,✉, Ana Kokeza 1, Barbara Soldo 3, Ivica Ljubenkov 3, Mara Banović 4, Danijela Skroza 1
PMCID: PMC6443773  PMID: 30996420

Abstract

Crljenak kaštelanski or Tribidrag is a Croatian autochthonous grape variety which is a parent of the America’s most popular variety Zinfandel or Italian Primitivo. The aim of this study was to investigate the dynamics of extraction of phenolics and changes in antioxidant activity of the samples collected during maceration of C. kaštelanski grapes processed using two different macerating enzymes. According to the obtained results it can be concluded that wines from C. kaštelanski grapes are great source of bioactive phenolics although differences in phenolic profiles between control and enzyme-treated wines were detected. The highest content of total phenolics was detected in control wine (2691 mg GAE/L). Use of pectolitic enzyme Vinozym Vintage improved anthocyanin extraction, while higher colour parameters were observed for wine samples produced using Sihazym Extro. The statistical analysis confirmed great influence of total phenolics and concentrations of some individual phenolic compounds (e.g. catechin, gallic acid, epicatechin) on reducing and free radical scavenging activity of wine samples while the impact of anthocyanins was not detected. According to the obtained results it can be concluded that use of enzymes has slightly negative effect on total phenolics and wine antioxidant properties, but it increases the extraction yield of wine colour components what enables shorter maceration and prevents colour losses during the aging process.

Keywords: Red wine, Vinification, Maceration enzymes, Phenolics, Anthocyanins, Antioxidants

Introduction

Croatia has historical tradition of grape vine cultivation and wine production. At the beginning of the last century, 400 different grape varieties were cultivated in Croatia, but since then the number of native varieties drastically decreased (Maletić et al. 2015). In the last few years considerable efforts have been taken to increase the areas under vines, and special attention was given to the native varieties and their revitalization.

Among those old cultivars, autochthonous Dalmatian red grape variety Crljenak kaštelanski is significantly outspread in Kaštela wine region (Central and South Dalmatia) (Maletić et al. 2015; Zdunić et al. 2013). It has attracted a lot of interest since American-Croatian team of scientists among more than 150 different autochthonous Dalmatian vines, discovered that C. kaštelanski (also called Tribidrag or Pribidrag) has an identical genetic profile as American most popular grape variety Zinfandel or Italian Primitivo. In the last few years, thanks to the Zinfandel’s reputation, this almost vanished grape cultivar has become economically important and recognized by Croatian winemakers who started to cultivate it increasingly. While 15 years ago, only a dozen vines were found in Dalmatian vineyards, C. kaštelanski is today planted on more than 75 ha (Žulj Mihaljević et al. 2015).

Due to proven health benefits of grape phenolics, and because of their importance to wine organoleptic properties and stability, the maceration of red grapes is found to be a crucial step in winemaking. Today, consumers are increasingly demanding highly coloured wines with the added biological value, meaning that extraction of anthocyanins from grape skin must be as complete as possible to ensure high colour intensity and improved stability during the aging process (Jackson 2000; Mattivi et al. 2006; Bautista-Ortín et al. 2007; Romero-Cascales et al. 2008; Moreno Arribas and Polo 2009; González-Neves et al. 2016; Generalić Mekinić et al. 2016). As traditional fermentation leads to a maximal extraction of anthocyanins up to 60%, the use of macerating enzymes to facilitate their extraction is a common practice. Therefore, these extraction processes modify not only colour of the red wine but also its stability, taste and structure (Bautista-Ortín et al. 2007; Romero-Cascales et al. 2008). The aim of this study was to compare the effects of two different winemaking enzymes on the evaluation and extraction of phenolics, colour components and antioxidants from C. kaštelanski during the vinification.

Materials and methods

Chemicals and instruments

All used reagents and solvents were of adequate analytical grade. Spectrophotometric measurements were performed on a Specord 200 (Analytik Jena GmbH, Germany) while phenolic compounds were analysed by high-performance liquid chromatography (HPLC) system (Perkin Elmer, Walthamn, Massachusetts, USA) consisting of an autosampler, vacuum degasser, binary pump, UV/VIS detector and the Peltier column oven (all of Series 200).

Sample preparation

Grapes from Vitis Vinifera var. C. kaštelanski were hand-picked at the stage of technological maturity from the vineyard located in Kaštela (Dalmatia, Croatia). All vines were of the same age (approximately 7 years old) and their identity have been confirmed. Immediately after the harvest, grapes were transported to the winery and processed. For each experiment 100 kg of grapes was used.

Winemaking and sampling

In this study three different winemaking procedures were performed; the traditional procedure without use of enzymes (as a control) and two procedures with different enzymes, Vinozym Vintage® FCE (Novozymes A/S, Bagsvaerd, Denmark) and Sihazym Extro (Eaton Begerow Product Line, Langenlonsheim, Germany) (3 g/100 L). The grapes were destemmed and crushed with a MGM-940 crusher (MIO, Osijek, Croatia) and distributed into different fermentation tanks. After the homogenisation, samples were treated with the enzymes, potassium metabisulphite (10 g/100 L) and inoculated by dry active yeasts SIHA®, Aktiv Hefe 8 (Burgundy Yeast) (E. Begerrow GmbH & Co., Langenlonsheim, Germany) (10 g/100 L). The cap of grape solid was kept soaked using the mechanical barrier. The fermentation temperature ranged from 25 to 27 °C. The maceration lasted for 5 days and, once the alcoholic fermentation has been finished, the must has been devatted and grape pomace was pressed by the manual screw press. Free-run juice and the pressed one were combined. The wine was than sealed by the tank’s floating lid and by the paraffin oil. At the beginning, during the maceration (from day 1 to day 5) samples were taken every day and finally after the racks (approximately 40 and 160 days after the winemaking process started). The samples were kept at − 20 °C until the analysis.

Spectrophotometric analysis of total phenolics, anthocyanins and wine colour parameters

The content of total phenolics was determined by Folin-Ciocalteu method (Singleton and Rossi 1965) and the results are expressed as mg of gallic acid equivalents per litre (mg GAE/L).

The amount of monomeric anthocyanins was determined by bisulphite bleaching assay (Amerine and Ough 1980) and the results are expressed as mg of malvidin-3-glucoside equivalents per litre (mg M-3-gl/L).

The colour intensity (CI), hue (H) and chromatic structure [optical density (OD) at 420, 520 and 620 nm] were determined spectrophotometrically and calculated according to the equations described by Ribéreau-Gayon et al. (2006).

HPLC analysis of phenolic compounds

Separations, quantifications and identifications of the individual phenolics were performed by HPLC method. Prior to injection, wine samples were filtered through the syringe filters (0.45 µm membrane pore size) and adequately diluted.

The phenolic compounds were separated on an UltraAqueous C18 column (250 × 4.6 mm, 5 µm, Restek, Bellefonte, PA, USA) maintained at 25 °C. The elution solvents were 0.2% phosphoric acid in water (solvent A) and 50% acetonitrile in methanol (solvent B). A gradient was applied as follows: from 96% A at 0 min to 50% A at 40 min, to 40% A at 45 min, to 20% A at 50 min, to 20% A at 53 min, then from 20 to 96% A at 54 min, and maintaining 96% A for 11 min (65 min). The applied flow rate was 0.8 mL/min and the injection volume was 20 µL. The detection of phenolics was carried out at 280 nm and peaks were identified by comparing their retention times and absorption spectra with those acquired for corresponding standards.

The HPLC analysis of anthocyanins was performed according to the Fredotović et al. (2017) with some slight modifications. The separation was carried out on the Kinetex C18 core-shell column (150 × 4.60 mm, 5 µm, Phenomenex, Torrance, CA, USA). The temperature was set to 40 °C and the elution solvents were 0.3% perchloric acid in water (solvent A) and 0.3% perchloric acid in methanol (solvent B). The flow rate was 0.6 mL/min and the injection volume was 10 µL. The detection of anthocyanins was carried out at 520 nm. The separated peaks were identified by their retention times, while their concentrations were determined according to the standard curve obtained for malvidin 3-O-glucoside.

Antioxidant activity

The reducing activity was measured as ferric reducing antioxidant power (FRAP) as described by Benzie and Strain (1996). This method is based on the reduction of ferric to ferrous ions at low pH which causes the formation of a coloured ferrous-tripyridyltriazine complex. The absorbance of the samples was recorded at 593 nm and the final results are expressed in micromoles of Trolox equivalents per litre (µmol TE/L) (Katalinić et al. 2013).

The free radical-scavenging ability of the samples was determined using a stable 2,2-diphenyl-2-picrylhydrazyl radical (DPPH·) according to the procedure described by Katalinić et al. (2013) and Harlina et al. (2018). This method is based on the “quenching” reactions of antioxidants with DPPH resulting in discolouration of the purple coloured ethanol solution which is monitored at 517 nm, and the results are expressed as DPPH inhibition (in %).

Statistical analysis

Statistical analysis was performed using GraphPad InStat3 (GraphPad Software, San Diego, USA) and STATISTICA (Data Analysis Software System, v. 10, StatSoft Inc, Tulsa, OK, USA). Pearson’s correlation coefficient was used for determination of the relations between the variables and p value < 0.05 was considered statistically significant. Duncan´s multiple comparison test was used to determine the significant differences between group means in an analysis of variance setting. All data are expressed as mean ± standard deviation (SD).

Results and discussion

Anthocyanins are the main pigments of red wines, and their content primary depends on phenolic composition of grapes and the applied winemaking procedure. Maceration is traditionally used in red winemaking and its duration is a critical step for obtaining stabile wines with good colour that will withstand a maturation period. While short maceration results with wines that contain low amounts of anthocyanins and have weak colour, the prolonged periods give wines with poor and unstable colour characteristics (Jackson 2000; Bautista-Ortín et al. 2007). The use of macerating enzymes to facilitate the extraction of grape components is a common and well-known practice. Use of enzymes support the extraction, maximize juice yield, facilitate filtration and intensify the wine flavour and colour. The short vatting with the addition of pectolytic enzymes increases wine colour intensity, but the concentration of extracted tannins remains low what results with wines that taste less astringent (Zoecklein et al. 1995; Pardo et al. 1999; Bautista-Ortín et al. 2007; Romero-Cascales et al. 2008; Mojsov et al. 2010; Río Segade et al. 2015). According to the study of Romero-Cascales et al. (2008) enzymes accelerate the extraction of phenolic compounds from grape skin but, if maceration period is too long the control wine can reach similar values of colour intensity. Therefore, enzyme preparations are found to be more useful in short macerations. Numerous studies investigated the effects of different oenological practices and macerating enzyme additions on the yield of extracted phenolics (Bautista-Ortín et al. 2007; Mojsov et al. 2010; Soto Vázquez et al. 2010; Río Segade et al. 2015; González-Neves et al. 2016; Aguilar et al. 2016). Despite the initial expectations, contradictory results have been reported but they all improve knowledge about the importance of choosing the proper winemaking technique.

In our study, two commercial enzyme preparations were used; Vinozym Vintage and Sihazym Extro. According to the producer’s product technical sheets the declared enzyme in both preparations was polygalacturonase with the activity of 7.500 and 7.600 polygalacturonase units/g in VinozymVintage and Sihazym Extro, respectively. Sihazym Extro enzyme also contained further defined secondary activities such as arabinosidase and hemicellulases.

The evolution of total phenolics and monomeric anthocyanins in C. kaštelanski wine during the vinification with and without enzymes is shown in Fig. 1. As expected, all samples had the maximum content of total phenolics at the end of vinification after the second rack and the highest value (2691 mg GAE/L) was detected for the control wine. While for the control sample the continuous growth of phenolics during vinification was recorded, a slight decrease of these compounds was detected for samples produced by other two vinification procedures. The results obtained for monomeric anthocyanins were completely different. The majority of monomeric anthocyanins were extracted during first few days of maceration, while in all cases the concentration of these compounds decreased from first to second rack. The extremely high values were recorded for Vinozym Vintage sample at second and for Sihazym Extro sample at fifth day of maceration but they were lower in samples collected after the second rack. Dimitrovska et al. (2015) also noted significant increase of total anthocyanins which occurred between the first and second day of the fermentation but soon after the maximum values were reached, the concentration continually dropped. This decrease of monomeric anthocyanins could be due to reactions of anthocyanins and formation of copigments by making weak linkages with other phenolic compounds. Furthermore, it is well known that the increase of alcohol content during vinification causes alterations in the unions responsible for copigmentation products, and the final result is usually a decrease of anthocyanins as a consequence of their fixation on the solid parts, adsorption on yeast cell walls, precipitation in the form of colloidal material, degradation reactions and structural changes (Moreno Arribas and Polo 2009; Soto Vázquez et al. 2010). Finally, the highest content of monomeric anthocyanins was found in Vinozym Vintage wine (115 mg M 3-gl/L). Mojsov et al. (2010) also reported that samples produced by the use of preparations Vinozym Vintage showed a more intensive extraction of anthocyanins in comparison to the control sample. As can be seen from the results presented in Fig. 1, used enzyme preparations had different effect on the yield of extracted phenolics and anthocyanins what supports the results of previous studies (Mojsov et al. 2010; Soto Vázquez et al. 2010; Ortega-Heras et al. 2012). Ortega-Heras et al. (2012) used also Vinozym Vintage enzyme preparation in vinification of Mencía grapes and the results are opposite to those obtained in our study where enzyme treated samples contained higher amounts of phenolics and anthocyanins. However, Parley et al. (2001) investigated the effects of pre-fermentation enzyme maceration on extraction and colour stability in Pinot Noir wine and also obtained lower content of phenolics, especially monomeric anthocyanins, in control sample than in enzyme treated wine. In the literature there are also numerous other reports showing that use of pectolitic enzymes do not improve anthocyanin extraction or that it may even cause decrease of their concentration (Soto Vázquez et al. 2010; Sun et al. 2011), probably due to polymerisation reactions or glycosidase side activities which may cause hydrolysis of these compounds.

Fig. 1.

Fig. 1

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Changes of total phenols (a) and total anthocyanins (b) in Crljenak kaštelanski wine during the vinification without enzyme addition and with the addition of enzyme A (Vinozym Vintage) or enzyme B (Sihazym Extro). GAE- gallic acid equivalents, M-3-gl- malvidin 3-glucoside. Different letters (a–e/f) on a and (a–e/g) on b in superscripts denote statistically significant difference (p < 0.05) of total phenolics and total anthocyanins among samples of the same wine collected at different vinification stages

Tables 1, 2, and 3 shows the results for individual phenolic compounds in samples detected by HPLC: three phenolic acids, two flavonoids from the group of flavan-3-ols, flavonol quercetin and stilbene resveratrol. Gallic acid was found to be the dominant phenolic acid and its highest concentration was detected in wine produced by the addition of Vinozym Vintage (about twofold higher concentrations in comparison to the control wine and wine produced with Sihazym Extro). Soto Vázquez et al. (2011) also reported the significant increase of this compound in wine produced using enzymes and tannins. Among flavonoids, catechin was the most abundant what is also in agreement with other reports (Bautista-Ortín et al. 2007; Soto Vázquez et al. 2010; Xia et al. 2010), followed by its epimer. Beside different concentrations of detected phenolic compounds among control wine and enzyme-prepared wines, the significant variations among samples at different stages of winemaking were also observed. Most of these individual phenolic compounds were detected in study by Soto Vázquez et al. (2010) who also obtained higher extraction yields of these phytochemicals in samples produced by the addition of enzymes and tannins. The presence of trans-resveratrol was confirmed in all samples with concentration range from 0.51 mg/L in wine produced by the addition of Vinozym Vintage to 1.07 mg/L in wine produced by the addition of Sihazym Extro.

Table 1.

Phenolic composition (mg/L) of Crljenak kaštelanski young wine produced by classic vinification without addition of enzymes

Group Phenolic compound (mg/L) Day 1 Day 2 Day 3 Day 4 Day 5 Rack 1 Rack 2
PA Gallic acid 8.41 ± 0.04a 12.56 ± 0.18b 17.78 ± 0.33c 8.97 ± 0.05a 24.67 ± 6.12d 25.46 ± 3.90de 28.63 ± 0.04e
Protocatechuic acid 1.29 ± 0.12a 0.95 ± 0.02bc 1.38 ± 0.13a 1.05 ± 0.02c 1.18 ± 0.12d 0.85 ± 0.00b 0.91 ± 0.05b
p-hydroxybenzoic acid n.d. 1.95 ± 0.02a 3.36 ± 0.67b n.d. 4.38 ± 0.57c 4.53 ± 0.55c 4.74 ± 0.01c
F Catechin 15.77 ±0.05a 39.70 ± 0.24b 55.06 ± 0.51c 24.29 ± 7.22d 67.14 ± 7.34e 76.27 ± 8.06f 82.60 ± 0.18g
Epicatechin 18.54 ±2.67a 28.86 ± 0.28b 33.97 ± 0.24c 21.30 ± 0.41d 66.79 ± 2.65e 43.35 ± 3.49f 47.04 ± 0.53g
Quercetin 0.17 ± 0.02a 1.37 ± 0.01b 0.98 ± 0.18c n.d. 0.98 ± 0.07c 1.27 ± 0.02b 1.27 ± 0.03b
S Resveratrol 0.10 ± 0.02a 0.44 ± 0.05b 0.50 ± 0.05c 0.30 ± 0.03b 0.50 ± 0.32c 0.68 ± 0.01d 0.70 ± 0.02d
A Delphinidin-3-O-glucoside 1.80 ± 0.01a 5.35 ± 0.00b 4.62 ± 0.07c 4.60 ± 0.01c 3.84 ± 0.03d 5.88 ± 0.10e 7.49 ± 0.37f
Cyanidin-3-O-glucoside 0.82 ± 0.07a 1.24 ± 0.07b 1.19 ± 0.05b 1.35 ± 0.07b 1.24 ± 0.13b 1.51 ± 0.15c 1.74 ± 0.34d
Petunidin-3-O-glucoside 2.37 ± 0.04a 6.94 ± 0.01b 6.61 ± 0.23b 6.93 ± 0.18b 6.17 ± 0.36c 8.35 ± 0.52d 8.56 ± 0.40d
Peonidin-3-O-glucoside 3.78 ± 0.11a 8.95 ± 0.46b 8.61 ± 0.56b 9.77 ± 0.38c 8.88 ± 0.39b 11.59 ± 0.46d 12.30 ± 0.65e
Malvidin-3-O-glucoside 17.70 ± 0.15a 50.12 ± 0.38b 51.32 ± 1.05c 56.98 ± 0.36d 49.14 ± 1.15e 61.14 ± 0.36f 50.49 ± 0.15b
Delphinidin-3-O-acetylglucoside 4.22 ± 0.27a 4.61 ± 0.04b 4.08 ± 0.19a 3.51 ± 0.13c 4.18 ± 0.44a 2.88 ± 0.14d 2.92 ± 0.17d
Cyanidin-3-O-acetylglucoside 1.04 ± 0.00a 1.48 ± 0.03b 1.28 ± 0.02c 1.45 ± 0.02b 1.71 ± 0.10d 1.47 ± 0.02b 0.47 ± 0.02e
Petunidin-3-O-acetylglucoside 1.78 ± 0.01a 2.58 ± 0.10b 2.32 ± 0.04c 2.55 ± 0.05b 2.76 ± 0.02d 2.60 ± 0.23b 0.91 ± 0.07e
Peonidin-3-O-acetylglucoside 1.12 ± 0.00a 1.93 ± 0.00b 1.62 ± 0.01c 1.70 ± 0.00d 1.83 ± 0.02e 1.11 ± 0.01a 0.58 ± 0.05f
Petunidin-(6-O-caffeoyl)glucoside 0.76 ± 0.00a 1.22 ± 0.01b 1.00 ± 0.01c 0.83 ± 0.03d 0.68 ± 0.01e 0.51 ± 0.03f 0.57 ± 0.01g
Malvidin-3-O-acetylglucoside 1.15 ± 0.01a 3.17 ± 0.00b 3.22 ± 0.01b 3.63 ± 0.06c 3.09 ± 0.02d 3.49 ± 0.13e 2.53 ± 0.01f
Malvidin-(6-O-caffeoyl)glucoside 0.67 ± 0.02a 2.09 ± 0.02b 2.08 ± 0.01b 1.96 ± 0.02c 1.58 ± 0.09d 1.85 ± 0.01e 1.43 ± 0.02f
Cyanidin-(6-O-coumaryoyl)glucoside 0.18 ± 0.00a 0.91 ± 0.01b 0.78 ± 0.08c 0.66 ± 0.00d 0.54 ± 0.01e 0.80 ± 0.01c 0.48 ± 0.01f
Petunidin-(6-O-coumaryoyl)glucoside 0.47 ± 0.00a 0.78 ± 0.01b 0.64 ± 0.00c 0.67 ± 0.01d 0.67 ± 0.00d 0.79 ± 0.00e 0.36 ± 0.01f
Peonidin-3-(6-O-coumaroyl)glucoside 0.41 ± 0.01a 1.39 ± 0.01b 1.33 ± 0.05c 1.31 ± 0.01c 1.15 ± 0.01d 1.34 ± 0.01e 0.81 ± 0.02f
Malvidin-3-(6-O-coumaroyl)glucoside 2.02 ± 0.01a 6.94 ± 0.01b 6.47 ± 0.10c 6.32 ± 0.02d 5.13 ± 0.03e 6.25 ± 0.04f 3.98 ± 0.01g
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PA Phenolic acids, F flavonoids, S stilbenes, A anthocyanins, n.d. not detected

Table 2.

Phenolic composition (mg/L) of Crljenak kaštelanski young wine produced with the addition of enzyme Vinozym Vintage

Group Phenolic compound (mg/L) Day 1 Day 2 Day 3 Day 4 Day 5 Rack 1 Rack 2
PA Gallic acid 8.69 ± 0.12a 8.92 ± 0.03a 24.91 ± 0.89b 34.23 ± 0.14c 30.05 ± 0.18d 50.39 ± 0.30e 65.45 ± 0.11f
Protocatechuic acid 0.92 ± 0.02a 0.93 ± 0.02ab 1.00 ± 0.03b 1.18 ± 0.04c 0.85 ± 0.11d 1.25 ± 0.08c 1.67 ± 0.02e
p-hydroxybenzoic acid 1.82 ± 0.01a 1.10 ± 0.00b 4.65 ± 0.07c 5.35 ± 0.05d 5.34 ± 0.02d 6.59 ± 0.16e 6.20 ± 0.07f
F Catechin 23.06 ± 2.80a 19.41 ± 0.29b 70.92 ± 0.52c 88.91 ± 0.15d 75.60 ± 0.52e 87.88 ± 0.98d 91.99 ± 0.10f
Epicatechin 23.84 ± 1.20a 22.26 ± 2.13b 45.12 ± 0.37c 56.00 ± 1.30d 45.60 ± 0.64b 48.21 ± 0.89e 52.85 ± 0.38f
Quercetin 0.34 ± 0.08a 0.12 ± 0.03b 0.52 ± 0.01c 0.65 ± 0.02d 0.43 ± 0.00e 1.41 ± 0.01f 2.18 ± 0.01g
S Resveratrol 0.39 ± 0.04a 0.32 ± 0.02b 0.92 ± 0.00c 0.91 ± 0.05c 0.67 ± 0.04d 0.86 ± 0.01e 0.51 ± 0.00f
A Delphinidin-3-O-glucoside 2.06 ± 0.03a 5.21 ± 0.25b 4.34 ± 0.22c 3.85 ± 0.04d 2.77 ± 0.01e 3.20 ± 0.05f 2.62 ± 0.01e
Cyanidin-3-O-glucoside 0.79 ± 0.01a 2.45 ± 0.05b 1.52 ± 0.29c 1.33 ± 0.08d 0.93 ± 0.05a 0.95 ± 0.01d 0.56 ± 0.03e
Petunidin-3-O-glucoside 2.83 ± 0.41a 8.69 ± 0.12b 6.78 ± 0.48c 6.47 ± 0.21c 4.67 ± 0.18d 5.68 ± 0.01e 4.03 ± 0.01f
Peonidin-3-O-glucoside 5.91 ± 0.52a 13.05 ± 0.03b 12.07 ± 1.40c 9.96 ± 0.23d 7.61 ± 0.23e 8.54 ± 0.02f 5.37 ± 0.13a
Malvidin-3-O-glucoside 20.89 ± 0.32a 63.48 ± 0.86b 63.12 ± 0.70b 57.82 ± 0.82c 46.12 ± 0.08d 51.82 ± 0.56e 36.29 ± 0.10f
Delphinidin-3-O-acetylglucoside 7.71 ± 0.20a 11.79 ± 0.15b 6.94 ± 0.46c 4.54 ± 0.49d 3.00 ± 0.02e 2.05 ± 0.05f 1.53 ± 0.01g
Cyanidin-3-O-acetylglucoside 1.25 ± 0.12a 1.22 ± 0.04a 1.02 ± 0.31b 1.09 ± 0.24ab 0.90 ± 0.01b 0.94 ± 0.02b 0.67 ± 0.00c
Petunidin-3-O-acetylglucoside 2.28 ± 0.03a 1.99 ± 0.04b 1.78 ± 0.31cd 1.82 ± 0.13c 1.65 ± 0.01d 1.72 ± 0.02cd 1.47 ± 0.02e
Peonidin-3-O-acetylglucoside 1.76 ± 0.22a 2.02 ± 0.02b 1.72 ± 0.12ac 1.64 ± 0.03c 1.48 ± 0.01d 0.99 ± 0.02e 0.58 ± 0.01f
Petunidin-(6-O-caffeoyl)glucoside 1.27 ± 0.08a 2.67 ± 0.25b 1.59 ± 0.24c 0.95 ± 0.19d 0.62 ± 0.14e 0.49 ± 0.00ef 0.35 ± 0.01f
Malvidin-3-O-acetylglucoside 1.28 ± 0.08a 4.25 ± 0.10b 4.39 ± 0.12b 3.59 ± 0.31c 2.81 ± 0.24d 3.37 ± 0.05e 2.17 ± 0.01f
Malvidin-(6-O-caffeoyl)glucoside 0.71 ± 0.07a 2.28 ± 0.03b 2.18 ± 0.02c 1.70 ± 0.01d 1.17 ± 0.01e 1.50 ± 0.02f 0.83 ± 0.04g
Cyanidin-(6-O-coumaryoyl)glucoside 0.17 ± 0.00a 0.73 ± 0.01b 0.62 ± 0.01c 0.57 ± 0.01d 0.38 ± 0.00e 0.47 ± 0.04f 0.29 ± 0.00g
Petunidin-(6-O-coumaryoyl)glucoside 0.52 ± 0.00a 0.62 ± 0.01b 0.48 ± 0.03c 0.50 ± 0.01c 0.49 ± 0.00c 0.66 ± 0.00d 0.74 ± 0.01e
Peonidin-3-(6-O-coumaroyl)glucoside 0.39 ± 0.06a 1.71 ± 0.05b 1.56 ± 0.01c 1.49 ± 0.10d 1.00 ± 0.01e 1.23 ± 0.01f 0.81 ± 0.01g
Malvidin-3-(6-O-coumaroyl)glucoside 1.72 ± 0.18a 8.24 ± 0.16b 7.57 ± 0.03c 6.77 ± 0.07d 4.63 ± 0.00e 5.47 ± 0.06f 3.60 ± 0.01g
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PA Phenolic acids, F flavonoids, S stilbenes, A anthocyanins, n.d. not detected

Table 3.

Phenolic composition (mg/L) of Crljenak kaštelanski young wine produced with the addition of enzyme Sihazym Extro

Group Phenolic compound (mg/L) Day 1 Day 2 Day 3 Day 4 Day 5 Rack 1 Rack 2
PA Gallic acid 5.38 ± 0.14a 7.19 ± 0.05a 9.21 ± 0.38a 14.45 ± 0.04b 23.75 ± 0.18c 44.58 ± 8.50d 45.04 ± 0.19d
Protocatechuic acid 1.99 ± 0.02a 0.56 ± 0.03b 0.59 ± 0.02b 1.61 ± 0.02ce 1.40 ± 0.08d 1.56 ± 0.36cd 1.78 ± 0.02e
p-hydroxybenzoic acid 1.83 ± 0.16a 1.63 ± 0.32ab 1.59 ± 0.05b 2.56 ± 0.02c 2.32 ± 0.20d 3.31 ± 0.01e 2.75 ± 0.01c
F Catechin 14.72 ± 0.15a 24.75 ± 0.19b 30.16 ± 0.47c 47.54 ± 0.57d 72.86 ± 0.61e 87.53 ± 1.48f 55.49 ± 3.93g
Epicatechin 10.82 ± 0.38a 19.60 ± 0.31b 18.93 ± 0.30c 24.99 ± 0.16d 34.75 ± 0.51e 48.65 ± 0.55f 22.03 ± 0.14g
Quercetin n.d. 0.71 ± 0.03a 0.43 ± 0.00b 0.67 ± 0.01a 1.48 ± 0.02c 1.34 ± 0.12d 5.19 ± 0.12e
S Resveratrol n.d. 0.35 ± 0.02a 0.44 ± 0.00b 0.68 ± 0.01c 0.89 ± 0.01d 0.90 ± 0.04d 1.07 ± 0.13e
A Delphinidin-3-O-glucoside 3.24 ± 0.01a 6.91 ± 0.00b 5.16 ± 0.07c 5.27 ± 0.04c 5.19 ± 0.17c 3.81 ± 0.35d 2.32 ± 0.01e
Cyanidin-3-O-glucoside 1.15 ± 0.04ac 1.30 ± 0.03b 1.01 ± 0.02a 1.23 ± 0.30bc 1.01 ± 0.05a 0.79 ± 0.08d 0.49 ± 0.00e
Petunidin-3-O-glucoside 3.79 ± 0.00a 8.96 ± 0.06b 7.23 ± 0.10c 7.81 ± 0.14d 8.18 ± 0.21e 5.60 ± 0.51f 3.04 ± 0.03g
Peonidin-3-O-glucoside 4.82 ± 0.02a 8.72 ± 0.11b 7.16 ± 0.13c 8.89 ± 0.13b 9.25 ± 0.04d 6.43 ± 0.46e 3.23 ± 0.03f
Malvidin-3-O-glucoside 24.73 ± 0.04a 58.74 ± 0.31b 50.07 ± 0.59c 61.55 ± 0.87d 63.82 ± 1.05e 40.58 ± 4.12f 21.63 ± 0.09g
Delphinidin-3-O-acetylglucoside 3.39 ± 0.00a 4.15 ± 0.25b 3.75 ± 0.06c 4.29 ± 0.15b 3.45 ± 0.17a 2.55 ± 0.19d 1.92 ± 0.05e
Cyanidin-3-O-acetylglucoside 1.05 ± 0.01ac 0.92 ± 0.02ab 0.90 ± 0.02b 1.19 ± 0.00c 1.67 ± 0.09d 1.37 ± 0.14e 1.86 ± 0.27f
Petunidin-3-O-acetylglucoside 2.05 ± 0.00a 1.76 ± 0.01b 1.68 ± 0.03b 2.35 ± 0.04c 2.73 ± 0.03d 2.48 ± 0.21e 2.96 ± 0.03f
Peonidin-3-O-acetylglucoside 1.11 ± 0.00ac 1.17 ± 0.00a 1.15 ± 0.01a 1.54 ± 0.16b 1.57 ± 0.09b 1.01 ± 0.11c 0.80 ± 0.00d
Petunidin-(6-O-caffeoyl)glucoside 0.67 ± 0.00a 1.26 ± 0.00b 0.87 ± 0.01c 1.01 ± 0.01d 0.99 ± 0.10d 0.60 ± 0.02e 0.48 ± 0.00f
Malvidin-3-O-acetylglucoside 1.26 ± 0.01a 3.27 ± 0.00b 2.52 ± 0.03c 3.44 ± 0.03d 3.58 ± 0.35d 2.13 ± 0.07e 1.04± 0.01f
Malvidin-(6-O-caffeoyl)glucoside 0.83 ± 0.01a 2.34 ± 0.09b 1.96 ± 0.03c 2.35 ± 0.01b 2.44 ± 0.18b 1.33 ± 0.14d 0.54 ± 0.00e
Cyanidin-(6-O-coumaryoyl)glucoside 0.34 ± 0.00a 1.15 ± 0.01b 0.84 ± 0.03c 0.93 ± 0.00d 0.89 ± 0.12cd 0.61 ± 0.08e 0.21 ± 0.00f
Petunidin-(6-O-coumaryoyl)glucoside 0.57 ± 0.00ac 0.54 ± 0.01a 0.45 ± 0.00b 0.61 ± 0.05c 0.75 ± 0.08d 0.80 ± 0.08d 1.16 ± 0.00e
Peonidin-3-(6-O-coumaroyl)glucoside 0.57 ± 0.02a 1.43 ± 0.00b 1.16 ± 0.01c 1.42 ± 0.03b 1.57 ± 0.07d 0.93 ± 0.07e 0.50 ± 0.00f
Malvidin-3-(6-O-coumaroyl)glucoside 3.24 ± 0.03a 8.92 ± 0.01b 6.90 ± 0.08c 7.91 ± 0.04d 7.95 ± 0.08d 4.92 ± 0.54e 2.24 ± 0.01f
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PA Phenolic acids, F flavonoids, S stilbenes, A anthocyanins, n.d. not detected

The qualitative and quantitative compositions of extracted anthocyanins are also shown in Tables 1, 2 and 3 where 16 different anthocyanin derivatives are presented. The dominant forms were those of 3-O-glucosides. Of these, malvidin is the most common pigment in Vitis vinifera varieties as well as the most stabile form of anthocyanins (Zoecklein et al. 1995; Río Segade et al. 2015; González-Neves et al. 2016). In wine produced without enzyme addition the share of 3-O-glucosides was 84% of total detected anthocyanins (95.60 mg/L), while in wines produced by the addition of enzymes it was 79% for Vinozym Vintage wine and 69% for Sihazym Extro, respectively. As expected, malvidin 3-O-glucoside was the dominant compound, followed by peonidin-3-glucoside, and its concentration in control wine was similar to those reported by Dimitrovska et al. (2015) for Merlot wine. Also, the concentrations of other monoglucosides in all samples were less abundant (lower than 10 mg/L). The concentration of other 3-O-glucosides was significantly higher in wine produced by the classic vinification in comparison to the wines produced with enzymes what is in accordance with results reported by Mattivi et al. (2006) for Primitivo (clone of C. kaštelanski) wine. Furthermore, changes in content of the individual anthocyanins during the maceration stages have been detected. The results for 3-O-glucosides in the control wine and wine produced with Vinozym Vintage showed concentration decrease of all detected 3-O-glucosides from second to fifth day of maceration what is in accordance with results obtained by Sun et al. (2011), while the application of Sihazym Extro caused increase of peonidin-3-O-glucoside (from 8.72 to 9.25 mg/L) and malvidin-3-O-glucoside (from 58.74 to 63.82 mg/L). Similar observations were obtained for O-coumaryoyl glucosides where increase of peonidins and petunidins was detected in wine produced using Sihazym Extro, while in all other cases decrease of these compounds was detected. In case of the control wine and Vinozym Vintage the content of 3-O-glucosides continued growth, the samples of wine produced using Sihazym Extro showed significantly lower concentrations. While Sun et al. (2011) in their study reported further decrease of all investigated anthocyanins at second rack (after 6 months), the results obtained in our study show similar trend for enzyme treated wines while the contents of all 3-O-glucosides except malvidin-3-O-glucoside in control wine were higher in samples from the second rack. An extremely significant correlations were found between the results for total monomeric anthocyanins and malvidin-3-O-glucoside (n = 21, r = 0.8973, p < 0.0001) and total malvidins (n = 21, r = 0.9078, p < 0.0001).

Figure 2 presents the distribution of anthocyanin derivatives in C. kaštelanski wine during the vinification with and without enzyme addition. As can be seen, the highest content of total delphinidins was detected in control wine what is in accordance with results reported by González-Neves et al. (2016). There were no significant differences among the detected concentrations of cyanidins in all samples, while the content of petunidins during the vinification without enzymes increased almost 2-times. Peonidins increased in classic vinification (from 5.31 to 13.68 mg/L), while slight decrease was detected in enzyme-treated wines. In all cases, the most significant increase of malvidin derivatives was detected from first to second day of maceration and the highest concentrations were detected in the control wine (58.43 mg/L).

Fig. 2.

Fig. 2

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Distribution of anthocyanin derivatives in Crljenak kaštelanski wine during the vinification without enzyme addition (a) and with Vinozym Vintage (b) and Sihazym Extro (c)

Colour parameters for C. kaštelanski wines produced by classic vinification and by the addition of enzymes are shown in Table 4. CI represents the amount of colour while T indicates the development of a colour towards orange. These parameters vary a great deal from one wine and grape variety to another (Ribéreau-Gayon et al. 2006). The most intensive colour was detected for wine produced by the use of Sihazym Extro (final CI value was 6.93), and the lowest CI was detected for wine produced by the addition of Vinozym Vintage. This is not in accordance with the results obtained by Mojsov et al. (2010) who, in their study on Vranec red wine produced with different pectolytic enzyme preparations (among which one of the used enzymes was Vinozym Vintage), reported higher CI values for wines produced using enzymes. T value for young wines usually ranges from 0.5 to 0.7 and it increases throughout aging, reaching an upper limit around 1.2–1.3 (Pardo et al. 1999; Ribéreau-Gayon et al. 2006) what is in accordance with our results. Colour composition is the contribution of each of the three basic colour components in the overall wine colour (Ribéreau-Gayon et al. 2006), and it can be defined as an optical density of the samples at 420 (yellow), 520 (red) and 620 (blue) nm, expressed in percentages. There were no significant differences between the OD 420 and OD 520 among samples produced without enzyme addition and with Vinozyme Vintage, while OD 420 was lower and OD 520 higher for wine produced with Sihazym Extro. The highest OD 620 was detected for the control sample (12.78%).

Table 4.

Colour parameters of Crljenak kaštelanski wine produced by classic vinification and by the addition of enzymes

Sample CI T Colour composition
OD 420 (%) OD 520 (%) OD 620 (%)
Classic vinification without enzyme addition
Day 1 2.71 0.54 32.50 60.44 7.06
Day 2 4.93 0.48 30.38 62.76 6.86
Day 3 4.65 0.51 31.25 61.71 7.04
Day 4 5.08 0.51 31.38 61.79 6.83
Day 5 5.32 0.52 31.91 61.06 7.03
Rack 1 5.66 0.55 32.67 59.55 7.77
Rack 2 6.18 0.76 37.64 49.59 12.78
Vinification with the addition of Vinozym Vintage enzyme
Day 1 2.65 0.56 33.12 59.59 7.29
Day 2 5.30 0.52 31.91 61.30 6.79
Day 3 4.52 0.54 32.79 60.20 7.01
Day 4 4.22 0.56 33.22 59.58 7.20
Day 5 3.70 0.58 34.12 58.54 7.35
Rack 1 4.51 0.68 36.55 53.98 9.47
Rack 2 5.38 0.74 38.19 51.45 10.36
Vinification with the addition of Sihazym Extro enzyme
Day 1 3.15 0.50 30.99 62.59 6.41
Day 2 4.01 0.46 29.77 64.42 5.81
Day 3 3.51 0.48 30.54 63.69 5.78
Day 4 4.86 0.48 30.63 63.48 5.89
Day 5 5.45 0.49 30.79 63.26 5.95
Rack 1 6.17 0.54 32.47 60.28 7.25
Rack 2 6.93 0.59 33.71 56.84 9.45
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CI Colour intensity; T hue; OD optical density

Although it is well-known that the phenolic composition of red wines varies significantly during vinification and storage, its effect on wine antioxidant activity has not been well elucidated (Sun et al. 2011). The antioxidant properties of investigated samples tested by two methods based on different mechanisms, FRAP and DPPH, are given in Fig. 3. All samples showed increase of reducing ability during the vinification. At the first day, the lowest FRAP value was detected for the control wine (3035 µmol TE/L), which at the end of winemaking (after second rack) showed the highest activity. Among other two samples, wine produced by the addition of Sihazym Extro had better antioxidant capacity. An extremely significant correlation has been confirmed between total phenolics and FRAP values (n = 21, r = 0.9773, p < 0.0001). Furthermore, the antioxidant capacity was determined by DPPH method. Again, extremely significant correlation has been confirmed between the results for total phenolic content and DPPH inhibition (n = 21, r = 0.9404, p < 0.0001). Other authors also observed a positive correlation between the phenolic content and wine antioxidant properties (López-Vélez et al. 2003; Katalinić et al. 2013; Paixão et al. 2007; Mitić et al. 2014). According to the obtained results it can be concluded that the antioxidant capacity of wine is largely influenced by the total phenolic content, while anthocyanins play a minor role (Fernández-Pachón et al. 2006; Cimino et al. 2007; Li et al. 2009; Mitić et al. 2014). In this study the antioxidant activity also didn’t show correlation with anthocyanins what can be explained by their chemical structure which is not catechol-type what is one of the most characteristic structure antioxidant features (Sun et al. 2011; Skroza et al. 2016). On the other hand, the correlation between tested antioxidant activities and content of detected individual compounds was also tested. The reducing activity (FRAP values) showed extremely significant correlation with catechin content (n = 21, r = 0.7204, p = 0.0002) and very significant correlation with the concentration of gallic acid (n = 21, r = 0.6591, p = 0.0012) and epicatechin (n = 21, r = 0.5483, p = 0.0101). Also results for DPPH inhibition correlated very significant with catechin content (n = 21, r = 0.5522, p = 0.0094) and significant with gallic acid (n = 21, r = 0.4946, p = 0.0227). Red wine contains a wide variety of phenolic substances and we investigated only few of them, so the influence of other compounds whose presence was not investigated should not be neglected. For example, Cimino et al. (2007) reported that the proanthocyanidin level has strong impact on the antioxidant efficiency of red wines, while according to the Fernández-Pachón et al. (2006) main contributors to a sherry and white wine radical scavenging activity are gallic, caffeic and caftaric acids and (−)-epigallocatechin gallate.

Fig. 3.

Fig. 3

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Comparison of the antioxidant properties of Crljenak kaštelanski wine samples collected during the vinification with and without enzymes obtained by FRAP (a) and DPPH (b) method. Enzyme A– Vinozym Vintage, Enzyme B– Sihazym Extro. Different letters (a–g) on Fig 3a and (a–d/e) on b in superscripts denote statistically significant difference (p < 0.05) of FRAP and DPPH inhibition values among samples of the same wine collected at different vinification stages

Conclusion

This study aimed to compare the classic vinification with those performed using macerating enzymes in order to investigate the phenolic potential of the grapes as well as the extraction of phenolics during the winemaking. Although there were differences in the extraction kinetic of phenolics between control and enzyme-treated wines during maceration, at the end the highest yield of phenolics was detected in control wine produced without enzymes. According to the obtained results it can be concluded that use of enzymes increases extraction yield of anthocyanins and other wine colour components what enables shorter maceration and prevents their losses during wine aging. Generally, the obtained results for phenolic profile and antioxidant properties of C. kaštelanski grapes imply that the content of total phenolics and some individual phenolic compounds (especially those with catechol type structure) is primarily responsible for antioxidant activity of the samples, while anthocyanins play a minor role. Generally, the obtain results are conformation that C. kaštelanski grapes are a great raw material for the production of high-quality, stabile and highly coloured wines with added biological value.

Acknowledgements

This work has been partially supported by Croatian Science Foundation under the project IP-2013-11-8652. We thank Prof. Vida Šimat for assistance with statistical analysis and suggestions that greatly improved the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.

Footnotes

Publisher's Note

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Contributor Information

Ivana Generalić Mekinić, Phone: +385 21 329 458, Email: gene@ktf-split.hr.

Živko Skračić, Phone: +385 21 329 458, Email: zivko.skracic@st.htnet.hr.

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