Yeast Yeast 2015; 32: 271–279. Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/yea.3060
Special Issue Article
Raspberry wine fermentation with suspended and immobilized yeast cells of two strains of Saccharomyces cerevisiae Radovan Djordjević1, Brian Gibson2, Mari Sandell3,8, Gustavo M. de Billerbeck4,5,6, Branko Bugarski7, Ida leskošek-Čukalović1, Jovana Vunduk1, Ninoslav Nikićević1 and Viktor Nedović1* 1
Department of Food Technology and Biochemistry, University of Belgrade, Serbia VTT Technical Research Centre of Finland, Espoo, Finland 3 University of Turku, Functional Foods Forum, Turku, Finland 4 Université de Toulouse, INSA, UPS, INP LISBP, Toulouse, France 5 INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France 6 CNRS, UMR5504, Toulouse, France; INP-ENSAT, Castanet-Tolosan Cedex, France 7 Department of Chemical Engineering, University of Belgrade, Serbia 8 University of Turku, Department of Biochemistry, Turku, Finland 2
*Correspondence to: V. Nedović, University of Belgrade, Faculty of Agriculture, Department of Food Technology and Biochemistry, Nemanjina 6, 11080 Zemun, Belgrade, Serbia. E-mail: [email protected]
Received: 3 July 2014 Accepted: 17 November 2014
Abstract The objectives of this study were to assess the differences in fermentative behaviour of two different strains of Saccharomyces cerevisiae (EC1118 and RC212) and to determine the differences in composition and sensory properties of raspberry wines fermented with immobilized and suspended yeast cells of both strains at 15 °C. Analyses of aroma compounds, glycerol, acetic acid and ethanol, as well as the kinetics of fermentation and a sensory evaluation of the wines, were performed. All fermentations with immobilized yeast cells had a shorter lag phase and faster utilization of sugars and ethanol production than those fermented with suspended cells. Slower fermentation kinetics were observed in all the samples that were fermented with strain RC212 (suspended and immobilized) than in samples fermented with strain EC1118. Significantly higher amounts of acetic acid were detected in all samples fermented with strain RC212 than in those fermented with strain EC1118 (0.282 and 0.602 g/l, respectively). Slightly higher amounts of glycerol were observed in samples fermented with strain EC1118 than in those fermented with strain RC212. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: fermentation; raspberry wine; Saccharomyces cerevisiae; immobilization; aroma, flavour; GC–FID; sensory evaluation
Introduction In modern winemaking, specific yeast strains have been used to guarantee the desired product quality. Yeasts are the dominant micro-organisms responsible for wine production and define the character of the wine through their metabolic activity (Fleet, 2003). Use of selected yeast strains, Saccharomyces cerevisiae strains in particular, in commercial winemaking became popular at the beginning of Copyright © 2014 John Wiley & Sons, Ltd.
the 1980s. Today, there are a range of commercial starter cultures available on the market and wine production processes are mostly based on use of S. cerevisiae and related yeast strains that allow rapid and reliable fermentations, reduce the risk of sluggish or stuck fermentations and restrict microbial contamination (Valero et al., 2005). Yeast starter cultures that are selected for winemaking on the basis of previous research ensure consistent production of desirable wines. With few exceptions,
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wines fermented with selected yeasts strains are of higher quality than wines produced by native microflora (Fleet and Heard, 1993; Romano et al., 2003). Some authors have shown that aromatic profiles of single-culture fermentations were significantly different from mixed-culture fermentations performed with grape must at laboratory scale. Wines produced by spontaneous fermentation are richer in aroma compounds, in contrast to those produced by selected single Saccharomyces cultures, but in such cases there is also increased risk of stuck fermentation and off-flavour production (HenickKling et al., 1998; Rojas et al., 2001). In recent years there has been an upsurge of interest in the use of immobilized cells, due to the attractive technical and economic advantages of immobilized cell technology compared to the conventional free cell system (Nedović et al., 2011; Margaritis and Merchant, 1984). Many immobilization supports have been proposed for use in wine making, such as sodium alginate, calcium alginate, Kissiris, gluten pellets, delignified cellulosic materials, DEAE–cellulose and fruit pieces (Kourkoutas et al., 2004). Immobilized cell systems not only increase productivity and improve the economic efficiency of bioprocesses but also influence yeast metabolism and, consequently, wine aroma, taste and product quality (Dervakos and Webb, 1991; Bakoyianis et al., 1993; Bardi and Koutinas, 1994; Melzoch et al., 1994; Sipsas et al., 2009). Wine aroma is the result of a complex combination of components that gives each wine its distinctive character. It has been shown that the main fraction of the aroma compounds (acetates, ethyl esters, higher alcohols, fatty acids, ketones and aldehydes) are produced during fermentation (Herraiz et al., 1991). Temperature is one of the most influential factors affecting fermentation. The modern practice, in most winemaking regions, is to conduct white wine fermentations at 8–15 °C, because this gives more fruity wines. In this respect, it is important to increase the synthesis of fruit esters, such as isoamyl acetate, isobutyl and hexyl acetates (Bardi et al., 1996; Kourkoutas et al., 2004; Mallouchos et al., 2003). The production of wine from berry fruits has increased over the last few years, in contrast to cider and perry production from apples and pears, which has been carried out since the eighteenth century. Raspberries (Rubus idaeus L.) represent a potentially valuable source of polyphenolic compounds, particularly flavonoids such as anthocyanin pigments, which Copyright © 2014 John Wiley & Sons, Ltd.
give raspberries their characteristic colour. Besides polyphenolic compounds, raspberry juice contains specific acid and sugar contents (pH 3.2–3.6 and about 14.5° Brix) that make them suitable for fruitwine production (Duarte et al., 2010a, 2010b). Raspberries are also attractive due to high fruit yields, a long harvest season, resistance to root rot and machine harvest characteristics. Raspberry fruits which can not be used for consumption are used in the production of juices, jam, sweets, etc. In some regions, raspberry producers are looking for new uses for small and crushed raspberry fruits. One such alternative is wine (Duarte et al., 2010a, 2010b). The aim of this work was to study the fermentation characteristics of two strains of S. cerevisiae in raspberry juice by analysing the kinetics of fermentation and by analysing primary and secondary (fermentative) aroma compounds from raspberry juice/wine. A further objective was to establish the differences between raspberry wine fermented with immobilized and suspended yeast cells of these two strains, and to determine differences in sensorial properties of the wines obtained.
Materials and methods Fermentation trials Raspberry fruits of the Willamette variety (R. ideaus) were obtained from a farm in the vicinity of Aleksandrovac town, Serbia. The fruits were stored at 25 °C prior to processing. The raspberry juice was prepared manually by crushing and pressing the raspberry fruits. The initial °Brix value was 11.71 and the pH was 3.02. Fermentations were carried out in triplicate in 1000 ml glass fermentors containing 500 ml filtersterilized raspberry juice. Filtration was carried out through a 0.22 μm pore diameter filter. Fermentations were performed at 15 °C, using suspended and immobilized cells of two different strains of Saccharomyces cerevisiae (EC1118 and RC212, Lallemand, Canada). The initial inoculation level in all samples was approximately 106 cells/ml of medium. The fermentation process took ca. 64 h. Samples were taken at intervals during fermentation (after 0, 16, 24, 40, 56 and 64 h of fermentation). Samples were analysed for fermentation kinetics and were retained for GC and HPLC analysis. Yeast 2015; 32: 271–279. DOI: 10.1002/yea
Raspberry wine fermentation with suspended and immobilized yeast cells
Fermentation kinetics (pH, sugar utilization and alcohol production) were determined from centrifuged and degassed fermentation samples, using a density meter (DMA 5000 M, Anton Paar GmbH, Austria) with Alcolyzer ME and pH ME modules (Anton Paar GmbH, Austria). In total, five samples/batch were fermented: • IMMOEC1118; sample fermented with immobilized S. cerevisiae EC1118 yeast strain. • IMMORC212; sample fermented with immobilized S. cerevisiae RC212 yeast strain. • SUSPEC1118; sample fermented with suspended S. cerevisae EC1118 yeast strain. • SUSPRC212; sample fermented with suspended S. cerevisiae RC212 yeast strain. • Control; pure, filter-sterilized, non-inoculated raspberry juice.
Encapsulation procedure The alginate (ALGOGEL™ 3001, Cargill) was generously donated by PALCO (Šabac, Serbia). Alginate powder was dispersed in distilled water to produce solutions of 0.015 g/ml. Before addition to alginate solutions, lyophilized yeast cells of both strains were rehydrated in water at 38 °C for 15 min and then added to alginate gel, and the suspension of alginate gel and cells was mixed with a magnetic stirrer for 1 h. The alginate/cells suspension was extruded through a 0.7 mm blunt stainless steel needle, using a syringe pump (Pump 11, Harvard Apparatus, USA) under a constant flow rate of 70 ml/h. The spherical droplets were formed by the combined action of electrostatic force and gravity. Electrostatic potential (6.5 kV) was formed by an electrostatic encapsulation unit (VAR V1, Nisco Encapsulator, Switzerland). The collecting solution was calcium chloride (0.015 g/ml). The distance between the needle tip and the collecting solution was 2.5 cm. After extrusion, the beads were left in the collecting solution for 45 min, after which the beads were rinsed and left in physiological solution at 4 °C (Nedović et al., 2001).
GC analysis of aroma compounds Acetaldehyde, 1-propanol, 2-methylpropanol, 2methylbutanol, 3-methylbutanol, 2-phenylethanol, ethyl acetate, 2-phenylethyl acetate, 3-methylbutyl Copyright © 2014 John Wiley & Sons, Ltd.
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acetate, ethyl caproate, ethyl caprylate and ethyl decanoate were determined and quantified by headspace gas chromatography (GC), using a 60 m DBWAX ETR column (i.d. 0.32 mm), hydrogen as the carrier gas, and flame ionization detection. Samples were injected at 150 °C and the temperature programme was 14 min at 60 °C, then at 10 °C/min to 85 °C, 10 min hold at 85 °C, and at 60 °C/min to 150 °C. β-Ionone, α-ionone, β-damascenone, α-ionol, geraniol, linalool, benzyl alcohol and hexanoic acid were qualitatively determined with a GC– GC system consisting of an Agilent 6890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with a Leco Pegasus 4D timeof-flight mass spectrometer (Leco Corp., St. Joseph, MI, USA). Extraction of these volatile compounds from the headspace of the vials was performed using a Gerstel MPS autosampler (Gerstel GmbH, Mülheiman der Ruhr, Germany) with an agitator and an SPME fibre. A 10 m × 0.18 mm i.d. Rxi5 ms (Restek Corp., Bellefonte, PA, USA) column, film thickness 0.18 μm, was used as the first column, and a 1.50 m × 0.1 mm i.d. BPX-50 (SGE Analytical Science, Austin, TX, USA) column, film thickness 0.1 μm, as the second column. A phenyl methyl deactivated retention gap column (1.5 m × 0.53 mm i.d.) was installed in front of the first column. The injector and transfer line temperatures were both 260 °C. Oven temperature programme conditions were as follows: initial temperature, 40 °C for 3 min, programmed at 7 ° C/min to 100 °C, followed by 10 °C/min to 270 °C, where it remained for 2 min. The secondary oven was kept 20 °C above the primary oven throughout the chromatographic run. The modulator offset was +35 °C in relation to the primary oven. Helium was used as carrier gas at a constant flow of 1.2 ml/min. Modulation time was 4 s. Electron impact ionization was applied at 70 eV, and the mass range 45–700 amu with 100 spectra/s were measured. 0.5 ml raspberry wine samples were pipetted in 20 ml headspace vials. An extraction time of 30 min and an extraction temperature of 80 °C were used. All samples were kept at 80 °C for 1 min prior to extraction. The headspace was sampled using a 2 cm DVB/CAR/PDMS 50/30 μm fibre (Supelco, Bellefonte, PA, USA). The analytes were desorbed in the GC inlet at 260 °C for 2.5 min and the fibre was reconditioned for 5 min at 260 °C after each analysis. Yeast 2015; 32: 271–279. DOI: 10.1002/yea
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HPLC analysis Residual sugars, acetic acid, glycerol and ethanol level in the samples were measured by HPLC. Analyses were performed using the Dionex ICS 3000 ion chromatography system with an anion exchange Carbo Pac PA-1 column with a flow rate of 0.3 ml/min. Compounds were identified by the retention times of authentic standards and quantified using a standard curve.
Sensory analysis Four wine samples and one raspberry juice sample were evaluated at the sensory laboratory of Functional Foods Forum (ISO 8589, Compusense five) at University of Turku. Consumers (31 women and 10 men) from the Turku area were asked to evaluate the hedonic ratings for odour, appearance, mouthfeel and taste, using a nine-point verbal scale: 1, extremely unpleasant; 2, very unpleasant; 3, moderately unpleasant; 4, slightly unpleasant; 5, neither unpleasant or pleasant; 6, slightly pleasant; 7, moderately pleasant; 8, very pleasant; 9, extremely pleasant.
Statistical analysis Statistical analyses were performed using the statistical program MS Excel (Microsoft Office 2007 Professional), using factorial repeated measures ANOVA when appropriate. If significant by ANOVA, differences in the means were determined using Duncan’s multiple range tests (p < 0.05).
Results and discussion
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protective role of the immobilization support (calcium alginate gel). Support materials are known to act as a protective agent against the physicochemical effects of pH, temperature, solvents and even heavy metals (Kourkoutas et al., 2004; Nedovic et al., 2000; Bardi et al., 1996). The difference in fermentation kinetics between the samples fermented with these two yeast strains is more obvious. It is clear that the EC1118 yeast strain, immobilized or suspended, is more favourable for this kind of fermentation than RC212 yeast strain. A higher concentration of ethanol at the end of fermentation was detected in SUSPEC1118 and SUSPRC212 samples in comparison to IMMOEC1118 and IMMORC212 samples (Table 1). This is probably a consequence of a higher concentration of cells in samples fermented with immobilized cells, as a higher amount of nutrients is necessary for biomass growth in that case. The same effect was observed by other authors (Oliveira et al., 2011). A significantly higher amount of acetic acid was detected in IMMORC212 and SUSPRC212 samples than in IMMOEC1118 and SUSPEC1118 (Table 1). Since the conditions of fermentation were the same in all samples, this result was most likely due to the influence of the yeast strain. A slightly higher (but statistically significant) concentration of glycerol was observed in IMMOEC1118 and SUSPEC1118 samples than in IMMORC212 and SUSPRC212 samples. Also, a higher concentration of glycerol was detected in samples fermented with suspended cells than with immobilized cells (Table 1). These results are contrary to those reported by Ciani and Ferraro (1996). The ratio between ethanol and glycerol content in samples was about 9:1.
Fermentation trials and kinetics of fermentation Fermentations lasted 64 h. In Figure 1, the kinetics of fermentations of samples are presented. Samples fermented with immobilized yeast cells (IMMOEC1118 and IMMORC212) started faster and had slightly faster utilization of sugars and ethanol production than those fermented with suspended cells (SUSPEC1118 and SUSPRC212). A faster start of fermentation and better fermentation rate in IMMOEC1118 and IMMORC212 samples were likely the consequence of improved stress tolerance of the immobilized cells, which is due to the Copyright © 2014 John Wiley & Sons, Ltd.
GC analysis of aroma compounds Acetaldehyde is the major carbonyl compound found in wine and other alcoholic beverages, with a wide range of concentrations (10–75 mg/l). Aldehydes contribute to flavour, with aroma perceived as ’bruised apple’ and ’nutty’, but are also a sign of wine oxidation. The most rapid accumulation of acetaldehyde during fermentation occurs during the time when the rate of carbon dissimilation is at its maximum, and decreases afterwards to a lower level by the end of fermentation. During Yeast 2015; 32: 271–279. DOI: 10.1002/yea
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Figure 1. Kinetics of fermentation of raspberry wine samples Table 1. Concentrations of ethanol, glycerol and acetic acid in the samples (g/l) Samples IMMOEC1118 SUSPEC1118 IMMORC212 SUSPRC212 CONTROL
Ethanol
Glycerol a
34.05 ± 0.13 37.13 ± 0.16b 33.96 ± 0.22a c 36.37 ± 0.11 0.00 ± 0.00d
Acetic acid a
3.70 ± 0.22 3.81 ± 0.14a 3.42 ± 0.16b b 3.56 ± 0.25 0.56 ± 0.33c
a
0.29 ± 0.10 0.28 ± 0.09a 0.44 ± 0.13b b 0.41 ± 0.17 0.11 ± 0.08c
1
Values are mean ± SD (n = 3). Values with different superscripts within the same columns are significantly different (p < 0.05).
a–d
wine storage and maturation, the level of this compound slowly increases. Fermentation conditions, such as medium composition, temperature of fermentation, oxygen presence and yeast strain, greatly affect acetaldehyde concentrations (Fleet, 2003; Swiegers et al., 2005). Copyright © 2014 John Wiley & Sons, Ltd.
Concentrations of acetaldehyde in samples IMMORC212 and SUSPRC212 were 36.3 and 41.7 mg/l, respectively. The concentrations in IMMOEC1118 and SUSPEC1118 samples were significantly lower, 27.9 and 28.7 mg/l, respectively. The concentration of acetaldehyde is dependent on the biocatalyst used. Samples fermented with suspended yeast cells showed a higher concentration of this compound compared to samples fermented with immobilized yeast cells. Ethyl esters are important fermentative aromatic compounds in wine, and amounts of these compounds are influenced by the yeast strain used, the temperature of fermentation, oxygen concentration and sugar content. These compounds positively influence overall wine quality and typically have a fruity aroma that contributes to the fruity and floral sensory properties of the wines (Swiegers et al., 2005). Samples IMMOEC1118 and IMMORC212 showed, respectively, 0.04 mg/l Yeast 2015; 32: 271–279. DOI: 10.1002/yea
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and 0.02 mg/l of ethyl decanoate (Table 2); in samples SUSPEC1118 and SUSPRC212, concentrations of this compound were two-fold lower. Duarte et al. (2010a, 2010b) reported that raspberry wines produced with S. cerevisiae CAT-1 and UFLA FW 15 contained 0.06 and 0.07 mg/l, respectively. Non-acetate esters, such as ethyl caproate and ethyl caprylate, can play an important role in wine aroma. Concentrations of these ethyl ester compounds were low, in the range 0.04–0.09 mg/l, but in agreement with data reported by other authors (Rojas et al., 2001). There were no connections between the biocatalyst used and the concentrations of these compounds found in the samples of raspberry wines. The synthesis of acetate esters by the yeast during fermentation is assisted by three acetyltransferases: alcohol acetyltransferase, ethanol acetyltransferase and isoamylalcohol acetyltransferase. These compounds contribute to the banana, apple, honey, rose and sweet aroma profile of wines (Lilly et al., 2000). Raspberry wine fermented with S. cerevisiae EC1118, both immobilized and suspended, showed the highest concentration of acetates. The most abundant compound among these was ethyl acetate (Table 2); the concentration was highest in the IMMOEC1118 sample and it decreased in the following order: IMMOEC1118 > SUSPEC1118 > IMMORC212 > SUSPRC212, depending on the biocatalyst used. A higher level of ethyl acetate was found in samples fermented with immobilized
cells than in samples fermented with suspended cells of both strains. In this group of compounds, 3-methylbutyl acetate (banana aromatic notes) was the compound that showed the biggest difference between samples; values of 0.38 and 0.27 mg/l were detected in samples IMMOEC1118 and SUSPEC1118, respectively (Table 2). In samples IMMORC 212 and SUSPRC212, the levels of this compound were significantly lower, 0.21 and 0.07 mg/l, respectively. 2-Phenylethyl acetate (apple, honey, rose, sweet) was found to range from 0.02 mg/l (SUSPRC212) to 0.23 mg/l (IMMOEC1118). Duarte et al. (2010a, 2010b) reported similar data for the level of this compound in raspberry wine samples, 0.19– 0.25 mg/l, depending on yeast strain. As shown in Table 2, the level of 3-methylbutyl acetate and 2-phenylethyl acetate in samples is strongly influenced by yeast strain and immobilization. An almost three-fold higher concentration of 2-phenylethyl acetate was detected in samples fermented with immobilized yeast cells. The same effect was observed by other authors: samples fermented with immobilized yeast cells showed higher concentrations of acetate esters in comparison to those fermented with suspended yeast cells (Mallouchos et al., 2003). Higher alcohol formation is dependent on the fermentation temperature, amongst other factors. An increase in fermentation temperature resultes in an increased concentration of total alcohols. Some authors have reported that higher alcohols
Table 2. Concentrations of volatiles in the samples (mg/l) Samples Compounds Acetaldehyde 1-Propanol 2-Methylpropanol 2-Methylbutanol 3-Methylbutanol 2-Phenylethanol Ethyl acetate 2-Phenylethyl acetate 3-Methylbutyl acetate Ethyl caproate Ethyl caprylate Ethyl decanoate 1
IMMOEC1118
SUSPEC1118
IMMORC212
SUSPRC212
27.86 ± 1.04a a 26.59 ± 0.53 15.59 ± 0.38a 9.90 ± 0.05a,b a 49.90 ± 0.33 30.22 ± 3.40a 13.76 ± 0.45a 0.23 ± 0.14a 0.38 ± 0.02a a 0.09 ± 0.00 0.06 ± 0.02a 0.04 ± 0.00a
28.74 ± 0.34a a 24.79 ± 1.25 15.91 ± 0.37a 10.57 ± 0.37a a 51.05 ± 2.58 12.64 ± 1.33b 12.51 ± 0.24a 0.09 ± 0.00b 0.27 ± 0.05a,b a 0.08 ± 0.02 0.09 ± 0.00b 0.03 ± 0.00b
36.28 ± 2.20b b 17.39 ± 1.47 18.58 ± 0.43b 7.99 ± 0.17b,c b 29.18 ± 2.50 7.97 ± 2.60c 10.51 ± 0.61b 0.06 ± 0.01c 0.21 ± 0.04b a 0.09 ± 0.01 0.04 ± 0.00a 0.02 ± 0.00c
41.72 ± 2.33c c 20.85 ± 4.82 23.18 ± 3.90c 9.27 ± 2.12a,b b 27.72 ± 3.07 4.52 ± 0.16d 6.52 ± 2.37c 0.02 ± 0.01d 0.07 ± 0.02c b 0.04 ± 0.00 0.04 ± 0.00a 0.01 ± 0.00c
Values are mean ± SD (n = 3). Values with different superscripts within the same row are significantly different (p < 0.05).
a–d
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Yeast 2015; 32: 271–279. DOI: 10.1002/yea
Raspberry wine fermentation with suspended and immobilized yeast cells
increase with amino acid increase in the must (Bardi et al., 1997; Herraiz et al., 1989). Other authors reported that higher alcohols can have both positive and negative impacts on the aroma and flavour of wine, depending their concentration; they are considered favourable compounds when their total concentration is < 300 mg/l (RibéreauGayon et al., 2006; Swiegers et al., 2005). Among this group of compounds, four compounds in total were measured: 1-propanol, 2-methylpropanol, 2-methylbutanol and 3-methylbutanol. The compound with the highest level in raspberry wine was 3-methylbutanol (range 51.05 mg/l for SUSPEC1118 to 27.72 mg/l for SUSPRC212). The concentrations of this compound detected in raspberry wine samples are in agreement with the data reported for grape wine samples (Mallouchos et al., 2003). Generally,
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higher concentrations of higher alcohols were detected in samples fermented with strain EC1118 than in those fermented with strain RC212. There was little difference in concentration of higher alcohols in samples fermented with immobilized or suspended yeast cells. The same results have been reported by other authors: in wines produced by immobilized cells on delignified cellulosic material and free cells, the concentrations of total alcohols were almost identical (Mallouchos et al., 2003). Flavour character in fruit comes through interactions between sugars, organic acids and ca. 200 volatile compounds. Raspberry fruits contain a range of volatile compounds with concentrations which are influenced by genotype, environment and season. Of the 230 volatiles characterized in
Figure 2. Relative ratio of aroma compounds in the raspberry wine samples
Figure 3. Hedonic ratings (mean + SD) for wine samples and control (juice) (n = 41 subjects) Copyright © 2014 John Wiley & Sons, Ltd.
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raspberry, only 12 have been identified that give the characteristic aroma of raspberries: β-ionone, α-ionone, β-damascenone, α-ionol, geraniol, linalool, (Z)-3-hexenol, benzyl alcohol, acetoin, raspberry ketone and acetic and hexanoic acids. These compounds are produced from primary metabolites through several pathways during fruit growth and ripening (Hampel et al., 2007; Malowicki et al., 2008; Furdíkova et al., 2014). In Figure 2 relative ratios of β-ionone, α-ionone, β-damascenone, α-ionol, geraniol, linalool, benzyl alcohol and hexanoic acid are presented. These compounds were qualitatively determined in raspberry samples. As seen in Figure 2, the level of these compounds in the control sample (nonfermented raspberry juice) is much higher than in all other samples. At the end of fermentation, in all other samples, the concentrations of these compounds were almost two-fold lower than in the control sample (at the beginning of fermentation) and were quite similar. Hexanoic acid is an exception among these compounds. In samples IMMOEC1118 and SUSPEC1118, no decrease in the level of this compound was observed. It is clear that during fermentation, transformation of these compounds occurred. A possible explanation is given by Zoecklein et al. (1999): monoterpenes can undergo considerable fluctuations as a result of isomerization and/or breakdown. It is possible that some changes in the concentration of individual free monoterpenes resulted from biochemical rearrangement in addition to hydrolysis; linalool may be formed from nerol and/or geraniol, while α-terpineol may be generated by linalool, nerol and/or geraniol. With the exceptions of α-terpineol, linalool, nerol, geraniol and pyran linalool oxides, most heat-induced terpenes can be attributed to rearrangement products of polyols.
Sensory analysis In general, the samples were quite similar to the consumers in appearance, mouthfeel and taste, and the results (mean + SD) are shown in Figure 3. The scores were highest for appearance (range of means, 6.8–7.3) perceived with a sense of sight, and lowest to tactile mouthfeel (5.4–5.8). A significant difference between the samples was found in pleasantness of odour, where juice was less pleasant than sample IMMOEC1118 (p < 0.05). Most Copyright © 2014 John Wiley & Sons, Ltd.
of the consumers were drinking wine at least once a month. The range of hedonic scores varied a lot and there were individual differences between consumers, which is normal. The same samples were either very pleasant or unpleasant for different consumers.
Conclusions Slightly faster fermentation kinetics were observed in samples fermented with immobilized cells in comparison to those fermented with suspended yeast cells. According to the results obtained, it was evident that in terms of fermentative characteristics, the strain EC1118 is superior to the strain RC212. Higher concentrations of all quantified volatile compounds, except for acetaldehyde, were observed in samples fermented with strain EC1118, immobilized and suspended. Concentrations of these compounds among samples fermented with immobilized and suspended cells of the same strain were quite similar. However, there was no association between the chemical composition and sensory characteristics of the samples. Acknowledgements This research was supported in part by EU FP7 COST Action FA0907 BIOFLAVOUR (www.bioflavour.insa-toulouse.fr) and the Ministry of Education and Science, Republic of Serbia (Project No. III 46010).
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‘Meeker’ (Rubus Idaeus L.). J Agric Food Chem 56(15): 6648–6655. Margaritis A, Merchant FJA. 1984. Advances in ethanol production using immobilized cell systems. Crit Rev Biotechnol 2: 339–393. Melzoch K, Rychtera M, Habova V. 1994. Effects of immobilization upon the properties and behavior of Saccharomyces cerevisiae cells. J Biotechnol 32: 59–65. Nedovic VA, Duirex A, Van Nedrvelde L, et al. 2000. Continuous cider fermentation with co-immobilized yeast and Leuconostoc oenos cells. Enzyme Microb Technol 26: 834–839. Nedović VA, Obradović B, Leskošek-Čukalović I, et al. 2001. Electrostatic generation of alginate microbeads loaded with brewing yeast. Process Biochem 37: 17–22. Nedovic V, Kalusevic A, Manojlovic V, et al. 2011. An overview of encapsulation technologies for food applications. Proc Food Sci 1: 1806–1815. Oliveira MD, Pantoja L, Duarte WF, et al. 2011. Fruit wine produced from cagaita (Eugenia dysenterica DC) by both free and immobilised yeast cell fermentation. Food Res Int 44(7): 2391–2400. Ribéreau-Gayon P, Glories Y, Maujean A, Dubourdieu D. 2006. Alcohols and other volatile compounds. In Handbook of Enology: The Chemistry of Wine Stabilization and Treatments, vol. 2, Ribéreau-Gayon P, Glories Y, Maujean A, Dubourdieu D (eds). Wiley: Chichester; 51–61. Rojas V, Gil JV, Piñaga F, Manzanares P. 2001. Studies on acetate ester production by non-Saccharomyces wine yeasts. Int J Food Microbiol 70(3): 283–289. Romano P, Fiore C, Paraggio M, et al. 2003. Function of yeast species and strains in wine flavour. Int J Food Microbiol 86(1): 169–180. Sipsas V, Kolokythas G, Kourkoutas Y, et al. 2009. Comparative study of batch and continuous multi-stage fixed-bed tower (MFBT) bioreactor during wine-making using freeze-dried immobilized cells. J Food Eng 90(4): 495–503. Swiegers JH, Bartowsky EJ, Henschke PA, Pretorius IS. 2005. Yeast and bacterial modulation of wine aroma and flavour. Austral J Grape Wine Res 11(2): 139–173. Valero E, Schuller D, Cambon B, et al. 2005. Dissemination and survival of commercial wine yeast in the vineyard: large-scale, three-year study. FEMS Yeast Res 5(10): 959–969. Zoecklein BW, Hackney CH, Duncan SE, Marcy JE. 1999. Effect of fermentation, aging and thermal storage on total glycosides, phenol-free glycosides and volatile compounds of white Riesling (Vitis vinifera L.) wines. J Indust Microbiol Biotechnol 22(2): 100–107.
Yeast 2015; 32: 271–279. DOI: 10.1002/yea
Special Issue Article
Raspberry wine fermentation with suspended and immobilized yeast cells of two strains of Saccharomyces cerevisiae Radovan Djordjević1, Brian Gibson2, Mari Sandell3,8, Gustavo M. de Billerbeck4,5,6, Branko Bugarski7, Ida leskošek-Čukalović1, Jovana Vunduk1, Ninoslav Nikićević1 and Viktor Nedović1* 1
Department of Food Technology and Biochemistry, University of Belgrade, Serbia VTT Technical Research Centre of Finland, Espoo, Finland 3 University of Turku, Functional Foods Forum, Turku, Finland 4 Université de Toulouse, INSA, UPS, INP LISBP, Toulouse, France 5 INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France 6 CNRS, UMR5504, Toulouse, France; INP-ENSAT, Castanet-Tolosan Cedex, France 7 Department of Chemical Engineering, University of Belgrade, Serbia 8 University of Turku, Department of Biochemistry, Turku, Finland 2
*Correspondence to: V. Nedović, University of Belgrade, Faculty of Agriculture, Department of Food Technology and Biochemistry, Nemanjina 6, 11080 Zemun, Belgrade, Serbia. E-mail: [email protected]
Received: 3 July 2014 Accepted: 17 November 2014
Abstract The objectives of this study were to assess the differences in fermentative behaviour of two different strains of Saccharomyces cerevisiae (EC1118 and RC212) and to determine the differences in composition and sensory properties of raspberry wines fermented with immobilized and suspended yeast cells of both strains at 15 °C. Analyses of aroma compounds, glycerol, acetic acid and ethanol, as well as the kinetics of fermentation and a sensory evaluation of the wines, were performed. All fermentations with immobilized yeast cells had a shorter lag phase and faster utilization of sugars and ethanol production than those fermented with suspended cells. Slower fermentation kinetics were observed in all the samples that were fermented with strain RC212 (suspended and immobilized) than in samples fermented with strain EC1118. Significantly higher amounts of acetic acid were detected in all samples fermented with strain RC212 than in those fermented with strain EC1118 (0.282 and 0.602 g/l, respectively). Slightly higher amounts of glycerol were observed in samples fermented with strain EC1118 than in those fermented with strain RC212. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: fermentation; raspberry wine; Saccharomyces cerevisiae; immobilization; aroma, flavour; GC–FID; sensory evaluation
Introduction In modern winemaking, specific yeast strains have been used to guarantee the desired product quality. Yeasts are the dominant micro-organisms responsible for wine production and define the character of the wine through their metabolic activity (Fleet, 2003). Use of selected yeast strains, Saccharomyces cerevisiae strains in particular, in commercial winemaking became popular at the beginning of Copyright © 2014 John Wiley & Sons, Ltd.
the 1980s. Today, there are a range of commercial starter cultures available on the market and wine production processes are mostly based on use of S. cerevisiae and related yeast strains that allow rapid and reliable fermentations, reduce the risk of sluggish or stuck fermentations and restrict microbial contamination (Valero et al., 2005). Yeast starter cultures that are selected for winemaking on the basis of previous research ensure consistent production of desirable wines. With few exceptions,
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wines fermented with selected yeasts strains are of higher quality than wines produced by native microflora (Fleet and Heard, 1993; Romano et al., 2003). Some authors have shown that aromatic profiles of single-culture fermentations were significantly different from mixed-culture fermentations performed with grape must at laboratory scale. Wines produced by spontaneous fermentation are richer in aroma compounds, in contrast to those produced by selected single Saccharomyces cultures, but in such cases there is also increased risk of stuck fermentation and off-flavour production (HenickKling et al., 1998; Rojas et al., 2001). In recent years there has been an upsurge of interest in the use of immobilized cells, due to the attractive technical and economic advantages of immobilized cell technology compared to the conventional free cell system (Nedović et al., 2011; Margaritis and Merchant, 1984). Many immobilization supports have been proposed for use in wine making, such as sodium alginate, calcium alginate, Kissiris, gluten pellets, delignified cellulosic materials, DEAE–cellulose and fruit pieces (Kourkoutas et al., 2004). Immobilized cell systems not only increase productivity and improve the economic efficiency of bioprocesses but also influence yeast metabolism and, consequently, wine aroma, taste and product quality (Dervakos and Webb, 1991; Bakoyianis et al., 1993; Bardi and Koutinas, 1994; Melzoch et al., 1994; Sipsas et al., 2009). Wine aroma is the result of a complex combination of components that gives each wine its distinctive character. It has been shown that the main fraction of the aroma compounds (acetates, ethyl esters, higher alcohols, fatty acids, ketones and aldehydes) are produced during fermentation (Herraiz et al., 1991). Temperature is one of the most influential factors affecting fermentation. The modern practice, in most winemaking regions, is to conduct white wine fermentations at 8–15 °C, because this gives more fruity wines. In this respect, it is important to increase the synthesis of fruit esters, such as isoamyl acetate, isobutyl and hexyl acetates (Bardi et al., 1996; Kourkoutas et al., 2004; Mallouchos et al., 2003). The production of wine from berry fruits has increased over the last few years, in contrast to cider and perry production from apples and pears, which has been carried out since the eighteenth century. Raspberries (Rubus idaeus L.) represent a potentially valuable source of polyphenolic compounds, particularly flavonoids such as anthocyanin pigments, which Copyright © 2014 John Wiley & Sons, Ltd.
give raspberries their characteristic colour. Besides polyphenolic compounds, raspberry juice contains specific acid and sugar contents (pH 3.2–3.6 and about 14.5° Brix) that make them suitable for fruitwine production (Duarte et al., 2010a, 2010b). Raspberries are also attractive due to high fruit yields, a long harvest season, resistance to root rot and machine harvest characteristics. Raspberry fruits which can not be used for consumption are used in the production of juices, jam, sweets, etc. In some regions, raspberry producers are looking for new uses for small and crushed raspberry fruits. One such alternative is wine (Duarte et al., 2010a, 2010b). The aim of this work was to study the fermentation characteristics of two strains of S. cerevisiae in raspberry juice by analysing the kinetics of fermentation and by analysing primary and secondary (fermentative) aroma compounds from raspberry juice/wine. A further objective was to establish the differences between raspberry wine fermented with immobilized and suspended yeast cells of these two strains, and to determine differences in sensorial properties of the wines obtained.
Materials and methods Fermentation trials Raspberry fruits of the Willamette variety (R. ideaus) were obtained from a farm in the vicinity of Aleksandrovac town, Serbia. The fruits were stored at 25 °C prior to processing. The raspberry juice was prepared manually by crushing and pressing the raspberry fruits. The initial °Brix value was 11.71 and the pH was 3.02. Fermentations were carried out in triplicate in 1000 ml glass fermentors containing 500 ml filtersterilized raspberry juice. Filtration was carried out through a 0.22 μm pore diameter filter. Fermentations were performed at 15 °C, using suspended and immobilized cells of two different strains of Saccharomyces cerevisiae (EC1118 and RC212, Lallemand, Canada). The initial inoculation level in all samples was approximately 106 cells/ml of medium. The fermentation process took ca. 64 h. Samples were taken at intervals during fermentation (after 0, 16, 24, 40, 56 and 64 h of fermentation). Samples were analysed for fermentation kinetics and were retained for GC and HPLC analysis. Yeast 2015; 32: 271–279. DOI: 10.1002/yea
Raspberry wine fermentation with suspended and immobilized yeast cells
Fermentation kinetics (pH, sugar utilization and alcohol production) were determined from centrifuged and degassed fermentation samples, using a density meter (DMA 5000 M, Anton Paar GmbH, Austria) with Alcolyzer ME and pH ME modules (Anton Paar GmbH, Austria). In total, five samples/batch were fermented: • IMMOEC1118; sample fermented with immobilized S. cerevisiae EC1118 yeast strain. • IMMORC212; sample fermented with immobilized S. cerevisiae RC212 yeast strain. • SUSPEC1118; sample fermented with suspended S. cerevisae EC1118 yeast strain. • SUSPRC212; sample fermented with suspended S. cerevisiae RC212 yeast strain. • Control; pure, filter-sterilized, non-inoculated raspberry juice.
Encapsulation procedure The alginate (ALGOGEL™ 3001, Cargill) was generously donated by PALCO (Šabac, Serbia). Alginate powder was dispersed in distilled water to produce solutions of 0.015 g/ml. Before addition to alginate solutions, lyophilized yeast cells of both strains were rehydrated in water at 38 °C for 15 min and then added to alginate gel, and the suspension of alginate gel and cells was mixed with a magnetic stirrer for 1 h. The alginate/cells suspension was extruded through a 0.7 mm blunt stainless steel needle, using a syringe pump (Pump 11, Harvard Apparatus, USA) under a constant flow rate of 70 ml/h. The spherical droplets were formed by the combined action of electrostatic force and gravity. Electrostatic potential (6.5 kV) was formed by an electrostatic encapsulation unit (VAR V1, Nisco Encapsulator, Switzerland). The collecting solution was calcium chloride (0.015 g/ml). The distance between the needle tip and the collecting solution was 2.5 cm. After extrusion, the beads were left in the collecting solution for 45 min, after which the beads were rinsed and left in physiological solution at 4 °C (Nedović et al., 2001).
GC analysis of aroma compounds Acetaldehyde, 1-propanol, 2-methylpropanol, 2methylbutanol, 3-methylbutanol, 2-phenylethanol, ethyl acetate, 2-phenylethyl acetate, 3-methylbutyl Copyright © 2014 John Wiley & Sons, Ltd.
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acetate, ethyl caproate, ethyl caprylate and ethyl decanoate were determined and quantified by headspace gas chromatography (GC), using a 60 m DBWAX ETR column (i.d. 0.32 mm), hydrogen as the carrier gas, and flame ionization detection. Samples were injected at 150 °C and the temperature programme was 14 min at 60 °C, then at 10 °C/min to 85 °C, 10 min hold at 85 °C, and at 60 °C/min to 150 °C. β-Ionone, α-ionone, β-damascenone, α-ionol, geraniol, linalool, benzyl alcohol and hexanoic acid were qualitatively determined with a GC– GC system consisting of an Agilent 6890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with a Leco Pegasus 4D timeof-flight mass spectrometer (Leco Corp., St. Joseph, MI, USA). Extraction of these volatile compounds from the headspace of the vials was performed using a Gerstel MPS autosampler (Gerstel GmbH, Mülheiman der Ruhr, Germany) with an agitator and an SPME fibre. A 10 m × 0.18 mm i.d. Rxi5 ms (Restek Corp., Bellefonte, PA, USA) column, film thickness 0.18 μm, was used as the first column, and a 1.50 m × 0.1 mm i.d. BPX-50 (SGE Analytical Science, Austin, TX, USA) column, film thickness 0.1 μm, as the second column. A phenyl methyl deactivated retention gap column (1.5 m × 0.53 mm i.d.) was installed in front of the first column. The injector and transfer line temperatures were both 260 °C. Oven temperature programme conditions were as follows: initial temperature, 40 °C for 3 min, programmed at 7 ° C/min to 100 °C, followed by 10 °C/min to 270 °C, where it remained for 2 min. The secondary oven was kept 20 °C above the primary oven throughout the chromatographic run. The modulator offset was +35 °C in relation to the primary oven. Helium was used as carrier gas at a constant flow of 1.2 ml/min. Modulation time was 4 s. Electron impact ionization was applied at 70 eV, and the mass range 45–700 amu with 100 spectra/s were measured. 0.5 ml raspberry wine samples were pipetted in 20 ml headspace vials. An extraction time of 30 min and an extraction temperature of 80 °C were used. All samples were kept at 80 °C for 1 min prior to extraction. The headspace was sampled using a 2 cm DVB/CAR/PDMS 50/30 μm fibre (Supelco, Bellefonte, PA, USA). The analytes were desorbed in the GC inlet at 260 °C for 2.5 min and the fibre was reconditioned for 5 min at 260 °C after each analysis. Yeast 2015; 32: 271–279. DOI: 10.1002/yea
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HPLC analysis Residual sugars, acetic acid, glycerol and ethanol level in the samples were measured by HPLC. Analyses were performed using the Dionex ICS 3000 ion chromatography system with an anion exchange Carbo Pac PA-1 column with a flow rate of 0.3 ml/min. Compounds were identified by the retention times of authentic standards and quantified using a standard curve.
Sensory analysis Four wine samples and one raspberry juice sample were evaluated at the sensory laboratory of Functional Foods Forum (ISO 8589, Compusense five) at University of Turku. Consumers (31 women and 10 men) from the Turku area were asked to evaluate the hedonic ratings for odour, appearance, mouthfeel and taste, using a nine-point verbal scale: 1, extremely unpleasant; 2, very unpleasant; 3, moderately unpleasant; 4, slightly unpleasant; 5, neither unpleasant or pleasant; 6, slightly pleasant; 7, moderately pleasant; 8, very pleasant; 9, extremely pleasant.
Statistical analysis Statistical analyses were performed using the statistical program MS Excel (Microsoft Office 2007 Professional), using factorial repeated measures ANOVA when appropriate. If significant by ANOVA, differences in the means were determined using Duncan’s multiple range tests (p < 0.05).
Results and discussion
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protective role of the immobilization support (calcium alginate gel). Support materials are known to act as a protective agent against the physicochemical effects of pH, temperature, solvents and even heavy metals (Kourkoutas et al., 2004; Nedovic et al., 2000; Bardi et al., 1996). The difference in fermentation kinetics between the samples fermented with these two yeast strains is more obvious. It is clear that the EC1118 yeast strain, immobilized or suspended, is more favourable for this kind of fermentation than RC212 yeast strain. A higher concentration of ethanol at the end of fermentation was detected in SUSPEC1118 and SUSPRC212 samples in comparison to IMMOEC1118 and IMMORC212 samples (Table 1). This is probably a consequence of a higher concentration of cells in samples fermented with immobilized cells, as a higher amount of nutrients is necessary for biomass growth in that case. The same effect was observed by other authors (Oliveira et al., 2011). A significantly higher amount of acetic acid was detected in IMMORC212 and SUSPRC212 samples than in IMMOEC1118 and SUSPEC1118 (Table 1). Since the conditions of fermentation were the same in all samples, this result was most likely due to the influence of the yeast strain. A slightly higher (but statistically significant) concentration of glycerol was observed in IMMOEC1118 and SUSPEC1118 samples than in IMMORC212 and SUSPRC212 samples. Also, a higher concentration of glycerol was detected in samples fermented with suspended cells than with immobilized cells (Table 1). These results are contrary to those reported by Ciani and Ferraro (1996). The ratio between ethanol and glycerol content in samples was about 9:1.
Fermentation trials and kinetics of fermentation Fermentations lasted 64 h. In Figure 1, the kinetics of fermentations of samples are presented. Samples fermented with immobilized yeast cells (IMMOEC1118 and IMMORC212) started faster and had slightly faster utilization of sugars and ethanol production than those fermented with suspended cells (SUSPEC1118 and SUSPRC212). A faster start of fermentation and better fermentation rate in IMMOEC1118 and IMMORC212 samples were likely the consequence of improved stress tolerance of the immobilized cells, which is due to the Copyright © 2014 John Wiley & Sons, Ltd.
GC analysis of aroma compounds Acetaldehyde is the major carbonyl compound found in wine and other alcoholic beverages, with a wide range of concentrations (10–75 mg/l). Aldehydes contribute to flavour, with aroma perceived as ’bruised apple’ and ’nutty’, but are also a sign of wine oxidation. The most rapid accumulation of acetaldehyde during fermentation occurs during the time when the rate of carbon dissimilation is at its maximum, and decreases afterwards to a lower level by the end of fermentation. During Yeast 2015; 32: 271–279. DOI: 10.1002/yea
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Figure 1. Kinetics of fermentation of raspberry wine samples Table 1. Concentrations of ethanol, glycerol and acetic acid in the samples (g/l) Samples IMMOEC1118 SUSPEC1118 IMMORC212 SUSPRC212 CONTROL
Ethanol
Glycerol a
34.05 ± 0.13 37.13 ± 0.16b 33.96 ± 0.22a c 36.37 ± 0.11 0.00 ± 0.00d
Acetic acid a
3.70 ± 0.22 3.81 ± 0.14a 3.42 ± 0.16b b 3.56 ± 0.25 0.56 ± 0.33c
a
0.29 ± 0.10 0.28 ± 0.09a 0.44 ± 0.13b b 0.41 ± 0.17 0.11 ± 0.08c
1
Values are mean ± SD (n = 3). Values with different superscripts within the same columns are significantly different (p < 0.05).
a–d
wine storage and maturation, the level of this compound slowly increases. Fermentation conditions, such as medium composition, temperature of fermentation, oxygen presence and yeast strain, greatly affect acetaldehyde concentrations (Fleet, 2003; Swiegers et al., 2005). Copyright © 2014 John Wiley & Sons, Ltd.
Concentrations of acetaldehyde in samples IMMORC212 and SUSPRC212 were 36.3 and 41.7 mg/l, respectively. The concentrations in IMMOEC1118 and SUSPEC1118 samples were significantly lower, 27.9 and 28.7 mg/l, respectively. The concentration of acetaldehyde is dependent on the biocatalyst used. Samples fermented with suspended yeast cells showed a higher concentration of this compound compared to samples fermented with immobilized yeast cells. Ethyl esters are important fermentative aromatic compounds in wine, and amounts of these compounds are influenced by the yeast strain used, the temperature of fermentation, oxygen concentration and sugar content. These compounds positively influence overall wine quality and typically have a fruity aroma that contributes to the fruity and floral sensory properties of the wines (Swiegers et al., 2005). Samples IMMOEC1118 and IMMORC212 showed, respectively, 0.04 mg/l Yeast 2015; 32: 271–279. DOI: 10.1002/yea
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and 0.02 mg/l of ethyl decanoate (Table 2); in samples SUSPEC1118 and SUSPRC212, concentrations of this compound were two-fold lower. Duarte et al. (2010a, 2010b) reported that raspberry wines produced with S. cerevisiae CAT-1 and UFLA FW 15 contained 0.06 and 0.07 mg/l, respectively. Non-acetate esters, such as ethyl caproate and ethyl caprylate, can play an important role in wine aroma. Concentrations of these ethyl ester compounds were low, in the range 0.04–0.09 mg/l, but in agreement with data reported by other authors (Rojas et al., 2001). There were no connections between the biocatalyst used and the concentrations of these compounds found in the samples of raspberry wines. The synthesis of acetate esters by the yeast during fermentation is assisted by three acetyltransferases: alcohol acetyltransferase, ethanol acetyltransferase and isoamylalcohol acetyltransferase. These compounds contribute to the banana, apple, honey, rose and sweet aroma profile of wines (Lilly et al., 2000). Raspberry wine fermented with S. cerevisiae EC1118, both immobilized and suspended, showed the highest concentration of acetates. The most abundant compound among these was ethyl acetate (Table 2); the concentration was highest in the IMMOEC1118 sample and it decreased in the following order: IMMOEC1118 > SUSPEC1118 > IMMORC212 > SUSPRC212, depending on the biocatalyst used. A higher level of ethyl acetate was found in samples fermented with immobilized
cells than in samples fermented with suspended cells of both strains. In this group of compounds, 3-methylbutyl acetate (banana aromatic notes) was the compound that showed the biggest difference between samples; values of 0.38 and 0.27 mg/l were detected in samples IMMOEC1118 and SUSPEC1118, respectively (Table 2). In samples IMMORC 212 and SUSPRC212, the levels of this compound were significantly lower, 0.21 and 0.07 mg/l, respectively. 2-Phenylethyl acetate (apple, honey, rose, sweet) was found to range from 0.02 mg/l (SUSPRC212) to 0.23 mg/l (IMMOEC1118). Duarte et al. (2010a, 2010b) reported similar data for the level of this compound in raspberry wine samples, 0.19– 0.25 mg/l, depending on yeast strain. As shown in Table 2, the level of 3-methylbutyl acetate and 2-phenylethyl acetate in samples is strongly influenced by yeast strain and immobilization. An almost three-fold higher concentration of 2-phenylethyl acetate was detected in samples fermented with immobilized yeast cells. The same effect was observed by other authors: samples fermented with immobilized yeast cells showed higher concentrations of acetate esters in comparison to those fermented with suspended yeast cells (Mallouchos et al., 2003). Higher alcohol formation is dependent on the fermentation temperature, amongst other factors. An increase in fermentation temperature resultes in an increased concentration of total alcohols. Some authors have reported that higher alcohols
Table 2. Concentrations of volatiles in the samples (mg/l) Samples Compounds Acetaldehyde 1-Propanol 2-Methylpropanol 2-Methylbutanol 3-Methylbutanol 2-Phenylethanol Ethyl acetate 2-Phenylethyl acetate 3-Methylbutyl acetate Ethyl caproate Ethyl caprylate Ethyl decanoate 1
IMMOEC1118
SUSPEC1118
IMMORC212
SUSPRC212
27.86 ± 1.04a a 26.59 ± 0.53 15.59 ± 0.38a 9.90 ± 0.05a,b a 49.90 ± 0.33 30.22 ± 3.40a 13.76 ± 0.45a 0.23 ± 0.14a 0.38 ± 0.02a a 0.09 ± 0.00 0.06 ± 0.02a 0.04 ± 0.00a
28.74 ± 0.34a a 24.79 ± 1.25 15.91 ± 0.37a 10.57 ± 0.37a a 51.05 ± 2.58 12.64 ± 1.33b 12.51 ± 0.24a 0.09 ± 0.00b 0.27 ± 0.05a,b a 0.08 ± 0.02 0.09 ± 0.00b 0.03 ± 0.00b
36.28 ± 2.20b b 17.39 ± 1.47 18.58 ± 0.43b 7.99 ± 0.17b,c b 29.18 ± 2.50 7.97 ± 2.60c 10.51 ± 0.61b 0.06 ± 0.01c 0.21 ± 0.04b a 0.09 ± 0.01 0.04 ± 0.00a 0.02 ± 0.00c
41.72 ± 2.33c c 20.85 ± 4.82 23.18 ± 3.90c 9.27 ± 2.12a,b b 27.72 ± 3.07 4.52 ± 0.16d 6.52 ± 2.37c 0.02 ± 0.01d 0.07 ± 0.02c b 0.04 ± 0.00 0.04 ± 0.00a 0.01 ± 0.00c
Values are mean ± SD (n = 3). Values with different superscripts within the same row are significantly different (p < 0.05).
a–d
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Raspberry wine fermentation with suspended and immobilized yeast cells
increase with amino acid increase in the must (Bardi et al., 1997; Herraiz et al., 1989). Other authors reported that higher alcohols can have both positive and negative impacts on the aroma and flavour of wine, depending their concentration; they are considered favourable compounds when their total concentration is < 300 mg/l (RibéreauGayon et al., 2006; Swiegers et al., 2005). Among this group of compounds, four compounds in total were measured: 1-propanol, 2-methylpropanol, 2-methylbutanol and 3-methylbutanol. The compound with the highest level in raspberry wine was 3-methylbutanol (range 51.05 mg/l for SUSPEC1118 to 27.72 mg/l for SUSPRC212). The concentrations of this compound detected in raspberry wine samples are in agreement with the data reported for grape wine samples (Mallouchos et al., 2003). Generally,
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higher concentrations of higher alcohols were detected in samples fermented with strain EC1118 than in those fermented with strain RC212. There was little difference in concentration of higher alcohols in samples fermented with immobilized or suspended yeast cells. The same results have been reported by other authors: in wines produced by immobilized cells on delignified cellulosic material and free cells, the concentrations of total alcohols were almost identical (Mallouchos et al., 2003). Flavour character in fruit comes through interactions between sugars, organic acids and ca. 200 volatile compounds. Raspberry fruits contain a range of volatile compounds with concentrations which are influenced by genotype, environment and season. Of the 230 volatiles characterized in
Figure 2. Relative ratio of aroma compounds in the raspberry wine samples
Figure 3. Hedonic ratings (mean + SD) for wine samples and control (juice) (n = 41 subjects) Copyright © 2014 John Wiley & Sons, Ltd.
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raspberry, only 12 have been identified that give the characteristic aroma of raspberries: β-ionone, α-ionone, β-damascenone, α-ionol, geraniol, linalool, (Z)-3-hexenol, benzyl alcohol, acetoin, raspberry ketone and acetic and hexanoic acids. These compounds are produced from primary metabolites through several pathways during fruit growth and ripening (Hampel et al., 2007; Malowicki et al., 2008; Furdíkova et al., 2014). In Figure 2 relative ratios of β-ionone, α-ionone, β-damascenone, α-ionol, geraniol, linalool, benzyl alcohol and hexanoic acid are presented. These compounds were qualitatively determined in raspberry samples. As seen in Figure 2, the level of these compounds in the control sample (nonfermented raspberry juice) is much higher than in all other samples. At the end of fermentation, in all other samples, the concentrations of these compounds were almost two-fold lower than in the control sample (at the beginning of fermentation) and were quite similar. Hexanoic acid is an exception among these compounds. In samples IMMOEC1118 and SUSPEC1118, no decrease in the level of this compound was observed. It is clear that during fermentation, transformation of these compounds occurred. A possible explanation is given by Zoecklein et al. (1999): monoterpenes can undergo considerable fluctuations as a result of isomerization and/or breakdown. It is possible that some changes in the concentration of individual free monoterpenes resulted from biochemical rearrangement in addition to hydrolysis; linalool may be formed from nerol and/or geraniol, while α-terpineol may be generated by linalool, nerol and/or geraniol. With the exceptions of α-terpineol, linalool, nerol, geraniol and pyran linalool oxides, most heat-induced terpenes can be attributed to rearrangement products of polyols.
Sensory analysis In general, the samples were quite similar to the consumers in appearance, mouthfeel and taste, and the results (mean + SD) are shown in Figure 3. The scores were highest for appearance (range of means, 6.8–7.3) perceived with a sense of sight, and lowest to tactile mouthfeel (5.4–5.8). A significant difference between the samples was found in pleasantness of odour, where juice was less pleasant than sample IMMOEC1118 (p < 0.05). Most Copyright © 2014 John Wiley & Sons, Ltd.
of the consumers were drinking wine at least once a month. The range of hedonic scores varied a lot and there were individual differences between consumers, which is normal. The same samples were either very pleasant or unpleasant for different consumers.
Conclusions Slightly faster fermentation kinetics were observed in samples fermented with immobilized cells in comparison to those fermented with suspended yeast cells. According to the results obtained, it was evident that in terms of fermentative characteristics, the strain EC1118 is superior to the strain RC212. Higher concentrations of all quantified volatile compounds, except for acetaldehyde, were observed in samples fermented with strain EC1118, immobilized and suspended. Concentrations of these compounds among samples fermented with immobilized and suspended cells of the same strain were quite similar. However, there was no association between the chemical composition and sensory characteristics of the samples. Acknowledgements This research was supported in part by EU FP7 COST Action FA0907 BIOFLAVOUR (www.bioflavour.insa-toulouse.fr) and the Ministry of Education and Science, Republic of Serbia (Project No. III 46010).
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