Physicochemical characterization of pomegranate wines fermented with three different Saccharomyces cerevisiae yeast strains

Accepted Manuscript Physicochemical characterization of pomegranate wines fermented with three different Saccharomyces cerevisiae yeast strains María Berenguer, Salud Vegara, Enrique Barrajón, Domingo Saura, Manuel Valero, Nuria Martí PII: DOI: Reference:

S0308-8146(15)00916-4 http://dx.doi.org/10.1016/j.foodchem.2015.06.027 FOCH 17709

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Food Chemistry

Received Date: Revised Date: Accepted Date:

16 February 2015 8 June 2015 9 June 2015

Please cite this article as: Berenguer, M., Vegara, S., Barrajón, E., Saura, D., Valero, M., Martí, N., Physicochemical characterization of pomegranate wines fermented with three different Saccharomyces cerevisiae yeast strains, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2015.06.027

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Physicochemical characterization of pomegranate wines fermented with three different Saccharomyces cerevisiae yeast strains

María Berenguer, Salud Vegara, Enrique Barrajón, Domingo Saura, Manuel Valero, Nuria Martí*

IBMC.-JBT Corp., FoodTech R&D Alliance, Instituto de Biología Molecular y Celular, Universidad Miguel Hernández (UMH)-Campus de Orihuela, Carretera de Beniel km 3.2, 03312 Orihuela, Alicante (Spain).

*Corresponding author. Tel: + 34 96 674 97 62; Fax: + 34 96 674 97 63; e-mail: [email protected] (N. Martí).

Abstract Three commercial Saccharomyces cerevisiae yeast strains: Viniferm Revelación, Viniferm SV and Viniferm PDM were evaluated for the production of pomegranate wine from a juice coupage of the two well-known varieties Mollar and Wonderfull. Further malolactic fermentation was carried out spontaneously. The same fermentation patterns were observed for pH, titratable acidity, density, sugar consumption, and ethanol and glycerol production. Glucose was exhausted while fructose residues remained at the end of alcoholic fermentation. A high ethanol concentration (10.91 ± 0.27% v/v) in combination with 1.49 g/L glycerol was achieved. Citric acid concentration increased rapidly a 31.7%, malic acid disappeared as result of malolactic fermentation and the lactic acid levels reached values between 0.40 g/L and 0.96 g/L. The analysis of CIEa parameter and total anthocyanin content highlights a lower degradation of monomeric anthocyanins during winemaking with Viniferm PDM yeast. The resulting wine retains a 34.5% of total anthocyanin content of pomegranate juice blend.

Keywords: Fermentation; Fruit wines; Wine yeast; Anthocyanins; Alcoholic beverage.

1. Introduction

Fermented beverages from fruit, or so-called "fruit wines" are made from various types of fruit: strawberry (Hidalgo et al., 2013), marula (Hiwilepo-van Hal et al., 2013), sand pear (Joshi et al., 2014), apple (Herrero et al., 1999), papaya (Lee et al., 2012), mango (Li et al., 2011, 2013), orange (Fernández-Pachón et al., 2014; Kelebek et al., 2009) and pomegranate (Mena et al., 2012; Zhuang et al., 2011). Fruit juice fermentation process follows more or less the same guidelines as those used for winemaking. During alcoholic fermentation, the fruit juice can suffer a number of variations that can be controlled. Depending on the fruit used, there are some parameters that will be important to keep the end consumer acceptance, such as color (Petravic-Tominac et al., 2013), aroma (Koppel et al., 2015; Molina et al., 2009) and distinctive taste plus others as its functional properties. The aroma and flavor profile of wine or fermented beverage is the result of an almost infinite number of variations in chemical and volatile composition, and generally, yeasts are the major contributor for modifying aroma, flavor, mouth-feel, color and chemical complexity. Initial characteristics of pomegranate juice used for fermented process vary depending on variety or selected blends (coupage) (Vegara et al., 2014). However this fruit is an ideal matrix for fermentation, due to that sugar content, an important factor for fermentation process, is in a concentration around 100-190 g/L in pomegranate juice (Ferrara et al., 2014; Türkyılmaz et al., 2013; Vegara et al., 2014). Pomegranate exhibit a wide range of biological properties that were attributed to the antioxidative properties of pomegranate polyphenols anthocyanins and hydrolysable tannins (such as punicalagins, punicalin, pedunculagin, gallagic and ellagic acid esters of glucose) (González-Molina et al., 2009; Mena et al., 2011). One characteristic of this

fruit is its red color, due to high concentrations of polyphenolic phytochemicals, particularly anthocyanin pigments. The predominant anthocyanins present in pomegranates are delphinidin 3,5-di (Dp3,5dG) and 3-O-glucoside (Dp3G), cyanidin 3,5-di (Cy3,5dG) and 3-O-glucoside (Cy3G), and pelargonidin 3,5-di (Pg3,5dG) and 3O-glucoside (Pg3G) (Vegara et al., 2013a). Unfortunately, anthocyanins are unstable and susceptible to degradation, leading to a brownish color during processing and storage (Vegara et al., 2013b). Therefore, the optimal fermentation of juice to maintain the qualities of the fruit would be one of the objectives required for the development of products derived from pomegranate. The purpose of this study was to compare the fermentation performance of a pomegranate juice blend by three commercial Saccharomyces cerevisiae yeast strains and the physicochemical composition of the resulting wines o beverages, especially the changes in pH, acidity, color, sugars and organic acids, anthocyanin content, and ethanol production.

2. Materials and Methods 2.1. Materials All chromatographic solvents were high-performance liquid chromatography (HPLC) grade. Formic acid and methanol were purchased from Scharlau (Scharlab, S.L., Sentmenat, Barcelona, Spain), and phosphoric acid from Panreac Química S.L.U. (Castellar del Vallès, Barcelona, Spain). Glucose, fructose and sucrose, citric, malic, acetic and lactic acids, as well as ethanol and glycerol standards were provided by Sigma-Aldrich

Química

S.A.

(Tres

Cantos,

Madrid

Spain);

anthocyanin

monoglucosides by Polyphenols Laboratories AS (Sandnes, Norway); and anthocyanin

diglucosides by Extrasynthese (Genay Cedex, France). Milli-Q water was produced using an Elix® Millipore water purification system (Molsheim, France).

2.2. Juice extraction Fruits from pomegranate varieties Mollar and Wonderfull were used in this study. Second quality pomegranate fruits harvested in autumn of 2013 were washed in cold tap water and drained. Then pomegranates were cut in halves and the outer leathery skin was removed. The arils were manually separated from the pith, placed inside a nylon mesh and pressed with a laboratory pilot press (Zumonat C-40; Somatic AMD, Valencia, Spain) as described by Vegara et al. (2013a). The extracted cloudy juice contained 2% pulp. Varietal juices were mixed in a ratio of 60:40 v/v (coupage).

2.3. Fermentation of pomegranate juice blend Three commercial wine S. cerevisiae yeasts (Agrovin S.A., Alcazar de San Juan, Ciudad Real, Spain): Viniferm Revelación (Yeast 1), Viniferm SV (Yeast 2) and Viniferm PDM (Yeast 3) were used in this study. Yeasts were grown separately in YPD broth (1% yeast extract, 2 % peptone and 2 % glucose, pH 6.5 ± 0.2) at 28 ºC for 48 h. Pre-cultures of the three Saccharomyces yeasts were used to inoculate pomegranate juice blends at a final concentration of 10 6 colony forming units (CFU)/mL. Bioreactors containing 50 L of pomegranate juice blend previously supplemented with 6 g/hL of potassium metabisulfite (Agrovin S.A.) were maintained at 22 ± 1 ºC during 35 days. Replicate pomegranate juice fermentations were carried out with each yeast strain. Samples were taken during fermentation at 0, 2, 9, 14, 22 and 32 days.

2.4. pH, titratable acidity and density

pH was measured at the indicated time points by using a pH-meter GLP 21 (Crison Instruments S.A., Alella, Barcelona, Spain). Titratable acidity (TA) and density were determined according to the International Federation of Fruit Juice Producers (Paris, France) IFU 1 and IFU 3 methods, respectively. TA was measured by titrating 10 g of pomegranate coupage or beverage with 0.5 mol/L NaOH up to pH 8.1 and results were expressed as g anhydrous citric acid (ACA) per kg (Vegara et al., 2014). Density (mg/L) was determined by using a pycnometer.

2.5 Color measurements The Minolta Chroma CR-300 (Minolta Co., Ltd., Chuo-Ku, Osaka, Japan) tristimulus colorimeter was used for color measurements at room temperature according to the CIE (Committee International d’Eclairage) Lab color notation system (McLaren, 1980). The instrument was calibrated with a white standard tile (L = 96.94, a = +0.18, b = +1.89). A glass Petri dish (52 mm diameter) containing pomegranate juice blend or fermented beverage was placed above a white tile and the CIELab values were determined (Giner et al., 2013). Hue angle (h) was calculated from tan−1 (b/a) and Chroma, color intensity or saturation (C) was calculated as (a 2 + b2)1/2.

2.5. Analysis of sugars, organic acids, ethanol and glycerol Samples were centrifuged at 15000xg for 5 min and then the supernatant was filtered through a 0.45 µm PVDF syringe filter. After that, it was passed through a solid phase separation C18 Sep-Pak cartridge (Waters Associates, Milford, MA, USA) preactivated with equal volumes of methanol, air and water (10:10:10, v/v). Organic acids and sugars were determined by HPLC (Mena et al., 2011; Molina et al., 2009; Santos et al., 2013) using a HP 1100 series system provided with an automatic injector and an

UV-detector, set at 210 nm wavelength, coupled with a refractive index detector (Hewlett-Packard, Palo Alto, CA, USA). A 10 µL sample was injected into a C610H Supelcogel (30 cm x 7.8 mm) column preheated at 30 °C and protected with a C610H Supelcogel (5 cm x 4.6 mm) guard column (Supelco, Bellefonte, PA, USA). The mobile phase consisted of 0.1 g/100 mL phosphoric acid at a flow of 0.5 mL/min. Glucose, fructose and sucrose, citric, malic, acetic and lactic acids, as well as ethanol and glycerol were characterized by chromatographic comparison with analytical standards and quantified by the area of their corresponding peaks. Calibration curves were obtained using different standard solutions. Results were expressed as g/L.

2.6. Determination of total anthocyanin content Quantitative analysis of anthocyanins was performed by HPLC (Vegara et al., 2013a) on a Model L6200 liquid chromatograph (Merck-Hitachi, Darmstadt, Germany) equipped with a SPD-M6A UV-VIS photodiode array detector (Shimadzu, Kyoto, Japan) and a Model 234 automatic sample injector (Gilson International Bv, Barcelona, Spain). Chromatograms were recorded and processed on a LC Workstation Class M10A Shimadzu PC-based chromatography data system. A 20 µL sample was analyzed on a Luna® 5 µm C18 column (25 × 0.46 cm) (Phenomenex Ltd., Macclesfield, UK) with a security guard cartridge system C18 ODS (4 × 3 mm), using a mobile phase of water/formic acid (95:5 v/v) (solvent A) and HPLC grade methanol (solvent B). Elution was performed at a flow rate of 1 mL/min. The linear gradient started with 1% B, keeping isocratic conditions during 5 min, reaching 20% B at 20 min, 40% B at 30 min, 95% B at 35 min and 1% B after 41 min. UV chromatograms were recorded at 520 nm and compared with those obtained with

analytical standards. Anthocyanins were quantified by the area of their corresponding peaks as Cy3G at 520 nm.

2.7. Statistical analysis Samples were analyzed in triplicate for each wine replicate and results expressed as the mean value ± standard deviation (SD). Statgraphics® Plus for Windows 3.0 (Statistical Graphic Corp. and Graphic Software Systems Inc., Rockville, Maryland, USA) was used for Analysis of Variance (ANOVA) and Fisher's least significant difference (LSD) procedure (P ≤ 0.05) to discriminate among the means.

3. Results and Discussion 3.1. pH, TA and density The three strains of S. cerevisiae yeasts had similar fermentations characteristics in terms of pH and density changes (Table 1). The pH values fluctuated from 3.40 to 3.58 and the density values from 952.30 to 997.60 mg/L for the three pomegranate wines analyzed. Pomegranate juice blend shows a density of 1024.05 ± 13.88 mg/L and decrease until day 22 of fermentation. No differences were found among pomegranate wines fermented with different yeast strains, but Yeast 3 produces a more rapid decrease than others (Figure 1B). Although in this study the density reduction was around 4.14%, other authors have reported both lower (2.94%) or similar (4.23%) values in orange and mandarin wines (Kelebelk et al., 2009; Selli et al., 2004). TA was followed during the fermentation process because it is easy to analyze the extent of increase or decrease of the different acids (citric, malic, lactic or acetic) that may be present. This parameter has a decisive influence on the aroma and flavor of fermented products and their levels can be used as an index of shelf-life. Initial TA

value for pomegranate juice blend was 2.81 ± 0.38 g citric acid/L and increased until day 9 of fermentation. From this moment, the TA level remained almost constant until the end of the study; nonetheless some fluctuations in its concentrations were found (Figure 1A). Wines fermented with the Yeasts 1 and 2 had a TA of 5.99 ± 0.06 g citric acid/L compared to 5.66 ± 0.14 g citric acid/L for that elaborated with the Yeast 3. Similar increases were found in other studies where juices of pomegranate, strawberry or orange were fermented (Andreu-Sevilla et al., 2013; Hidalgo et al., 2013; Kelebek et al., 2009). This increase in TA might be due to the production of α-ketoglutaric and succinic acids in the glyceropyruvic fermentation pathway during winemaking, especially at the beginning of the alcoholic fermentation (Ribéreau-Gayon et al., 2006). Pyruvic acid is derived from glycolysis and in glyceropyruvic fermentation it does not form ethanal and ethanol (the NADH is used to reduce dihydroxyacetone) and thus goes on to form secondary products, such as α-ketoglutaric acid, succinic acid, acetoin, diacetyl, 2,3-butanediol, etc.

3.2. Color evolution during fermentation Color of pomegranate juice blend and fermented beverage was evaluated using the CIELab system of chromatic coordinates, an objective tool for color perception. Results are presented in Figure 1C-G. Statistical differences were found for CIELab parameters between the pomegranate juice blend and wine (Table 1). From these, it is noteworthy that the wine fermented with the Yeast 3 showed the highest CIEa value that determines an intensity of red color only comparable to that of the pomegranate fresh juice blend. In addition, Yeast 3 produced wine with low CIEb value that indicates a limited presence of yellow constituents and minor h value. Color of pomegranate

fermented beverage is an important parameter for consumer acceptance (Mena et al., 2013).

3.3- Sugar consumption Glucose and fructose were the two reducing sugars detected in pomegranate juice blend (Figure 2A). Sucrose was not detected in pomegranate wines before and after fermentation. The initial glucose and fructose contents were 60.21 ± 0.57 g/L and 70.73 ± 3.34 g/L, respectively. Kinetic changes of fructose in pomegranate wines during fermentation with the three Saccharomyces yeasts are shown in Figure 3A. Yeasts 2 and 3 showed a similar pattern of fructose utilization. The fructose content in the juice blends inoculated with these yeasts displayed a rapid (lineal) reduction until day 14 and remained constant afterwards. The final contents of fructose were 4.05 ± 2.19 g/L and 5.11 ± 1.05 g/L for Yeast 2 and Yeast 3, respectively. During the 2 first days Yeast 1 did not consume fructose, but thereafter showed a rapid increase in the consumption of fructose until day 9. Then the concentration of fructose decreased slightly and remained constant after day 14, with 5.93 ± 3.63 g/L of fructose at the end of fermentation process. Yeast 1 showed the fastest consumption of glucose among the three yeasts (Figure 3B). At day 9, glucose consumption was complete in the fermentation process. The Yeasts 2 and 3 consumed glucose more slowly and depleted its content on day 14. The rapid decrease in sugar content and the consequent increase in the concentration of ethanol (Figure 3C) confirmed that the selected yeasts dominated the fermentation process. The glucose and fructose contents were drastically reduced during the alcoholic fermentation, finishing around day 14, and kinetic changes of these carbohydrates were very similar. Nonetheless, while glucose almost disappeared,

fructose residues remained at the end of the winemaking process (Figure 2B), as in mulberry (Kim et al., 2008), orange (Kelebek et al., 2009; Santos et al., 2013) and pomegranate (Mena et al., 2012) wines.

3.4. Organic acids during fermentation The major organic acid in pomegranate juices is citric acid in a concentration of 0.6-18.5 g/L, depending of variety (Mena et al., 2011; Vegara et al., 2014). In this work, three organic acids: citric, malic and lactic acids were separated and identified in the pomegranate juice blend and wines. The evolution of citric acid content during fermentation process is shown in Figure 4A. Citric acid concentration in pomegranate juice blend was 7.19 ± 0.61 g/L. This concentration increased rapidly to reach the maximum levels on day 9, then remaining more or less stable until the end of winemaking process with a final value of 9.47 ± 0.43 g/L. No differences were found among the three different yeasts used (Table 1). Citric acid content in fruit wines may increase (Chen and Liu, 2014; Mena et al., 2014) or decrease (Lee et al., 2012; Santos et al., 2013) during fermentation. Malic acid was the second most abundant organic acid in pomegranate juice blend and wines. It underwent noticeable changes during the winemaking process. The decreasing concentrations of malic acid in the wines (Figure 4B), compared with the coupage, could be attributed to malolactic fermentation because lactic acid was produced. Lactic acid is not present in sound fruits and derived juice; it is indicator of spoilage by lactic acid bacteria (LAB) (Vegara et al., 2014). The malic acid level in pomegranate juice blend was 5.60 ± 0.51 g/L and disappeared at the end of winemaking, as established by other authors (Mena et al., 2012; Santos et al., 2013).

During winemaking, species of LAB were developed towards the end of alcoholic fermentation or at later stages (Figure 4C). In fact, the occurrence of lactic acid was registered on day 14 in wines fermented with the Yeast 3 and on day 22 in those wines fermented with the Yeasts 1 and 2. The highest lactic acid formation was registered for wines fermented with Yeasts 1 and 2 at the end of winemaking process (on day 32), reaching concentrations of 0.93 ± 0.02 g/L and 0.67 ± 0.06 g/L respectively. According with these results, alcoholic and malolactic fermentations overlap slightly throughout winemaking and the applied level of sulfitation was unable to prevent LAB growth. Pomegranate juice blend lacked acetic acid and it was not registered during the winemaking process. Acetic acid is an undesirable organic acid in alcoholic beverages that imparts vinegary off-odour at concentrations near its flavour threshold: 0.7–1.1 g/L (Lambrechts and Pretorius, 2000).

3.5. Ethanol and glycerol production Ethanol contents of approximately 10.6%, 11.1% and 11.0% (v/v) were determined for pomegranate wines fermented with the three Saccharomyces Yeasts 1, 2 and 3, respectively (Table 1). These results are higher than those reported in other studies on pomegranate (between 8.30% and 9.05% v/v) (Andreu-Sevilla et.al 2013) and other fruit wines, such as marula (5.5% v/v) (Hiwilepo-van Hal et al., 2013) or mango (between 6.50% and 7.99% v/v) (Li et al., 2013; Reddy and Reddy, 2005). Nonetheless, the ethanol content of the pomegranates wines that were produced herein is comparable to that (10.7% v/v) reported in other study on mango wine (Pino and Queris, 2011). The highest ethanol concentration was achieved after 14 days of fermentation (Figure 3C).

Glycerol is a by-product generated by S. cerevisiae and a non-volatile alcohol with no aromatic properties, but it significantly contributes to wine quality by providing sweetness and fullness (Fleet and Heard, 1993). During alcoholic fermentation under anaerobic conditions, glycerol equilibrates the cytosolic redox balance and acts as an osmoregulatory metabolite in response to the high osmotic pressure of the sugar-rich juices (Wang et al., 2001). Although no glycerol was detected at day 0, its concentration increased drastically in only 2 days, reaching the highest concentration after 9 days of fermentation. The three selected yeast showed a similar pattern of glycerol production (Figure 3D) and final concentration of 1.49 ± 0.02 g/L.

3.6. Anthocyanin profile of pomegranate wines The anthocyanin profile is a critical parameter for the characterization of pomegranate juices. The fruit yields consistent patterns from different varieties and geographical origins (AIJN, 2012). The typical anthocyanin profile of pomegranate juices is characterized by delphinidin, cyanidin and pelargonidin 3-glucosides and 3,5diglucosides (Alighourchi et al., 2008; Martí, et al., 2002; Vegara et al., 2013a). The anthocyanin content plays a main role on the attractive color and the antioxidant capacity of pomegranate juice (Mena et al., 2013). Individual anthocyanin concentrations for Mollar and Wonderfull pomegranate juice blend (60:40) were, in decreasing order, Cy3G (175.45 ± 6 mg/L), followed by Cy3,5dG (162.31 ± 4.30 mg/L), Pg3G (30.08 ± 4.00 mg/L), Dp3,5dG (25.32 ± 0.90 mg/L), Dp3G (14.06 ± 1.20 mg/L) and Pg3,5dG (0.9 ± 0.10 mg/L). Winemaking process had notable influence on the degradation of individual anthocyanins as indicated by changes in their contents (Figure 5A-C). Irrespective of the yeast strain used to ferment the pomegranate juice blend, Cy3G and Cy3,5dG decreased slightly

throughout the alcoholic fermentation while negligible variations took place for the other individual anthocyanin values. After this stage of wine elaboration (14 days), a noticeable reduction was seen for all anthocyanins. The two majoritarian anthocyanins at the end of winemaking process were Cy3,5dG and Cy3G, with a concentration of 42.43 ± 5.20 and 51.57 ± 2.70 mg/L for the wine fermented with Yeast 1, 30.58 ± 5.50 and 63.90 ± 5.00 mg/L for the wine fermented with Yeast 2, and 71.99 ± 5.10 and 68.94 ± 5.2 mg/L for that fermented with Yeast 3. Higher stability of diglucoside anthocyanins than monoglucoside ones was observed by Alighourchi and Barzegar (2009) and Vegara et al. (2014). The decrease in individual anthocyanin concentrations might not be due to alcoholic and malolactic fermentations because anthocyanins are susceptible to degradation during storage at room temperature (Vegara et al., 2013a).

4. Conclusions The same fermentation patterns by the three commercial wine S. cerevisiae yeast strains Viniferm Revelación, Viniferm SV and Viniferm PDM were observed for pH, titratable acidity, density, sugar consumption, and ethanol and glycerol production. Glucose was exhausted while fructose residues remained at the end of alcoholic fermentation. A high ethanol concentration (10.91 ± 0.27% v/v) in combination with 1.49 g/L glycerol was achieved after 14 days of fermentation. Citric acid concentration increased a 31.7% during the 9 first days, remaining almost constant until the end of winemaking process (day 32); on the contrary, malic acid disappeared as result of malolactic fermentation. The lactic acid levels reached values between 0.40 g/L and 0.96 g/L. The analysis of CIEa parameter and total anthocyanin content in pomegranate wines highlights a lower degradation of monomeric anthocyanins during winemaking with Viniferm PDM yeast. This type of wine retains a 34.5% of total anthocyanin content of pomegranate juice blend. Results suggest that it may be possible to produce

pomegranate wines with improved characteristics by selecting the fermentative yeast strain. Saccharomyces cerevisiae var. bayanus [Prise de Mousse (PDM) strain] is cryophilic yeast capable of fermenting under conditions of nutrient deficiency that produces low quantities of aromatic compounds and respects the substrate typicity. Therefore, the combination of selected yeast strain and a lower temperature of fermentation (< 22 ºC) could help to reduce anthocyanin degradation in pomegranate wine, avoiding a dramatic impact on its color and preserving the beneficial effects of these specific bioactive compounds on human health.

Acknowledgements The authors wish to thank Program Becas Santander CRUE CEPYME, designed to complement the training of students in Spanish universities, approaching them the reality of the professional field, expanding their knowledge and fostering contact with companies, for the opportunity given to María Berenguer, Vitalgrana Pomegranate S.L. for providing plant material and Mitra Sol Technologies S.L. for the financial support of this work.

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Figure captions Figure 1. Evolution of titratable acidity (A), density (B) and CIEL (C), a (D), b (E), Chroma (F) and Hue angle (G) color parameters in pomegranate wines during fermentation by Saccharomyces cerevisiae Viniferm Revelación (●), Viniferm SV (■) and Viniferm PDM (▲).

Figure 2. Chromatographic profiles of main reducing sugars in pomegranate juice blend (A) as well as ethanol and glycerol production in pomegranate wine (B). Major components labeled: 1, glucose; 2, fructose; 3, glycerol; 4, ethanol.

Figure 3. Changes of fructose (A), glucose (B), ethanol (C) and glycerol (D) concentration in pomegranate wines during fermentation by Saccharomyces cerevisiae Viniferm Revelación (●), Viniferm SV (■) and Viniferm PDM (▲).

Figure 4. Changes of organic acids: citric (A), malic (B) and lactic (C) acid in pomegranate wines during elaboration process by using Saccharomyces cerevisiae Viniferm Revelación (●), Viniferm SV (■) and Viniferm PDM (▲).

Figure 5. Evolution of individual monomeric anthocyanins (● Dp3,5dG, ■ Cy3,5dG, ▲ Pg3,5dG, ▼ Dp3G, ♦ Cy3G, and ᴏ Pg3G) during winemaking process by using different Saccharomyces cerevisiae yeast strains: Viniferm Revelación (A), Viniferm SV (B) and Viniferm PDM (C).

Fig. 1

B

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Fig. 5

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Table 1. Physicochemical properties, organic acid, sugar, ethanol, glycerol and anthocyanin contents of pomegranate wines before and after fermentation. Day 0

Day 32 Viniferm Revelación

3.46 ± 0.02*a pH 1024.05 ± 13.88a Density (mg/L) 2.81 ± 0.38c Titratable acidity (g citric acid/L) 18.32 ± 0.08c CIE L 4.63 ± 0.03a CIE a -0.19 ± 0.02c CIE b 4.65 ± 0.03b Chroma (C) 7.79 ± 0.13c Hue angle (h) 0.00 ± 0.00 Ethanol (% v/v) 0.00 ± 0.00 Glycerol (g/L) 7.19 ± 0.61b Citric acid (g/L) 5.60 ± 0.51 Malic acid (g/L) 0.00 ± 0.00 Lactic acid (g/L) 70.73 ± 3.34a Fructose (g/L) 60.21 ± 0.57 Glucose (g/L) 411.17 ± 13.23a Total anthocyanins (mg/L) *

3.46 ± 0.02a 981.78 ± 11.92b 6.03 ± 0.15a 22.32 ± 1.61b 2.77 ± 0.79c 3.52 ± 0.72b 4.48 ± 1.05b 52.27 ± 2.63a 10.62 ± 1.14a 1.51 ± 0.10a 9.08 ± 0.64a 0.00 ± 0.00 0.93 ± 0.02a 5.93 ± 3.63b 0.00 ± 0.00 91.26 ± 12.81c

Viniferm SV 3.47 ± 0.01a 979.10 ± 15.99b 5.94 ± 0.10a 25.10 ± 0.22a 3.49 ± 0.08b 5.08 ± 0.11a 6.17 ± 0.08a 55.49 ± 1.03a 11.15 ± 0.96a 1.47 ± 0.09a 9.90 ± 1.32a 0.00 ± 0.00 0.67 ± 0.06b 4.05 ± 2.19b 0.00 ± 0.00 92.51 ± 12.73c

Viniferm PDM 3.49 ± 0.08a 984.20 ± 13.08b 5.66 ± 0.14b 23.59 ± 3.03ab 4.97 ± 0.64a 3.61 ± 1.38b 6.19 ± 1.32a 34.74 ± 7.19b 10.97 ± 1.23a 1.50 ± 0.11a 9.62 ± 0.59a 0.00 ± 0.00 0.50 ± 0.05c 5.19 ± 1.05b 0.00 ± 0.00 141.76 ± 4.73b

Values are means ± standard deviation (SD). Means in the same row followed by different letters are significantly different (P ≤ 0.05).

Highlights Three commercial Saccharomyces cerevisiae yeasts were investigated to ferment pomegranate juice ► Differences during fermentation process were evaluated with different yeasts ► Sugars, organic acids, ethanol, anthocyanin content and color parameters were determined ► It possible to produce pomegranate wines with improved characteristics by selecting the fermentative yeast strain