Abstract
Two autochthonous yeasts from the northwest region of Argentina, Kloeckera apiculata mc1 and Saccharomyces cerevisiae mc2, were used as pure or mixed starter cultures in microvinification trials conducted in Malbec red must. Also, the effect of Oenococcus oeni X2L was evaluated. S. cerevisiae mc2 showed adequate growth and fermentative activity in single and composite fermentations, producing standard concentration of ethanol. The amount of esters was higher in fermentations conducted using mixed yeast starters. Independent of the timing of inoculation of O. oeni, this malolactic bacterium completely depleted malic acid. Sensory evaluation indicated that young wines fermented with mixed yeast cultures and sequential inoculation of O. oeni were preferred, achieving the highest scores for positive descriptors and they allowed better control of the sensory quality. Consequently, this study proposes inclusion of autochthonous K. apiculata mc1 as an adjunct culture to S. cerevisiae mc2 during Malbec must fermentation to improve the organoleptic properties of red wines. Furthermore, sequential inoculation of O. oeni X2L should be carried out after completion of the alcoholic fermentation to enhance sensory characteristics.
Introduction
In the winemaking process, alcoholic fermentations (AF) are currently conducted using starters of selected strains of Saccharomyces cerevisiae in contrast to traditional spontaneous fermentations carried out by the flora present on the grapes and in the winery. Despite having advantages such as the easy control and homogeneity of fermentations, wines produced with S. cerevisiae monocultures have often shown lack of flavor complexity, stylistic distinction, and vintage variability [25, 45]. New fermentation technologies for optimizing wine quality and producing wines with particular flavor profiles is one of the worldwide trends in oenology [45]. The use of mixed starter cultures would permit one to improve wine quality, taking advantage of spontaneous fermentations without the risks of stuck fermentations or wine spoilage [6, 20, 43, 45]. Some non-Saccharomyces yeast species can improve the fermentation behavior of yeast starter cultures and lead to a more complex aroma [5, 9, 17, 44].
In a previous report we determined that K. apiculata mc1 in mixed cultures with S. cerevisiae mc2 in basal medium survive longer than in pure culture [32]. Despite the consistent production of secondary products by single culture of K. apiculata, an adequate production of these compounds was observed in mixed culture. The existence of K. apiculata mc1, autochthonous non-Saccharomyces yeast might be of technological interest in Argentinean winemaking. However, in wine biotechnology more specific information on the extent of its contribution is required.
In this regard it is important to know the fermentation behavior of these non-Saccharomyces yeasts in mixed culture with S. cerevisiae in regional musts. Also, in the red wine fermentation it is necessary to consider the inclusion of Oenococcus oeni, a bacterium mainly involved in malolactic fermentation (MLF). This fermentation improves the wine quality because acidity decreases, the microbiological stability increases, and the sensory characteristics are enhanced with additional flavors [7, 16]. This step is often difficult to accomplish because of the inadequate physico-chemical conditions after the AF [2, 3, 27, 38].
The aim of this study was to carry out a comparative analysis of chemical and sensory characteristics of Argentinean typical red wines fermented by pure or mixed cultures of two indigenous yeasts, K. apiculata mc1 and S. cerevisiae mc2. Also, the influence of O. oeni X2L on the wine quality was evaluated.
Materials and methods
Microorganisms and growth conditions
K. apiculata mc1 and S. cerevisiae mc2, previously isolated from Malbec grape musts (northwest region, Argentina) were used throughout the study. The two yeasts strains were chosen according to their kinetics and metabolic performance previously examined [32]. Yeast strains were subcultured at 6-month intervals in YPD (10 g/l yeast extract, 20 g/l peptone, 20 g/l glucose, pH 4.0) agar slants and maintained at 4°C. Yeast strains were propagated in YPD broth at 25°C for 24 h at least three times prior to experimental use.
O. oeni strain X2L, isolated from Malbec red wine (Cafayate, Argentina) was utilized [47]. This bacterium was kept frozen at −20°C in MRS (Man Rogosa Sharpe) broth containing 52 g/l MRS broth (Merck, Darmstadt, Germany) added with 20% glycerol (v/v). O. oeni was grown in MRS broth supplemented with 150 ml/l of natural tomato juice (pH 4.0) at 30°C for 24 h at least three times prior to experimental use.
All microorganisms were obtained from the wine yeasts and lactic acid bacteria (LAB) collection at the Microbiology Institute of the Biochemistry, Chemistry and Pharmacy Faculty (Tucumán National University, Argentina).
Preparation of starter cultures
Erlenmeyer flasks (100 ml) containing 80 ml of pasteurized grape juice, previously diluted 1:2 with sterile water and adjusted to pH 4.0, were inoculated with 2 ml of young cultures of K. apiculata mc1, S. cerevisiae mc2, or O. oeni X2L. Cultures were incubated at 28°C during 24 or 48 h for yeasts and bacterium, respectively. Subsequently, cells were collected by centrifugation at 8,000g for 10 min at 4°C and resuspended in 5 ml of sterile grape juice.
Fermentation conditions
The Malbec variety was utilized to conduct microvinification trials because it is the most typical red grape of Argentina. Grapes were harvested during the 2009 vintage.
Fermentations were carried out in 1-l Erlenmeyer flasks containing 800 ml of Malbec red must in the presence of skins (sugars 245 g/l, assimilable nitrogen 125 mg/l, titratable acidity 4.7 g/l, l-malic acid 1.9 g/l, pH 4.1) at 25°C. The grape must was exposed to heat treatment (80°C for 10 min) and subsequently the must was treated with 50 mg/l sulfur dioxide (as sodium metabisulfite). Concentration of the preservative was chosen because of its lack inhibitory effect on the yeasts and lactic acid bacterium employed in the fermentation trials.
The flasks were aseptically inoculated with different starter cultures to obtain an initial cell density of 5 × 106 cfu/ml for yeasts and/or malolactic bacterium.
Five inoculation strategies were applied: (1) pure culture of K. apiculata mc1; (2) pure culture of S. cerevisiae mc2; (3) mixed culture of both yeasts; (4) mixed culture of yeasts and simultaneous inoculation of O. oeni X2L; and (5) mixed culture of yeasts and sequential inoculation of bacterium after completion of the AF. Fermentations inoculated with pure cultures were carried out as controls.
After aseptic stoppering of the flasks with a special valve containing sulfuric acid to allow only CO2 to escape from the system [4], weight loss was monitored for several days until the end of the fermentation (constant weight for two consecutive days). Fermentations were carried out under static conditions.
Enumeration of microbiological populations
Populations of yeasts and bacterium were followed by counting viable cells. Differential enumeration of wine yeasts was performed by plating on the following selective media: (1) Malt agar medium (20 g/l glucose, 1 g/l peptone, 20 g/l yeast extract, 3 g/l malt extract, and 20 g/l agar, pH 6.8) that allows morphologically non-Saccharomyces yeast to be distinguished from Saccharomyces colonies. (2) Modified malt agar medium by addition of 40 mg/l bromophenol, pH 4.6; non-Saccharomyces yeast appears as blue colonies while Saccharomyces forms yellow ones. (3) Malt agar medium with the addition of cycloheximide at a concentration (1 mg/l) that is only inhibitory for the growth of Saccharomyces yeast [23, 32]. The plates were incubated at 28°C for 3–5 days.
O. oeni was enumerated by inoculating adequate dilutions onto the plates of MRS agar supplemented with cycloheximide at a concentration (100 mg/l) to inhibit the growth of both yeast genera. The plates were incubated at 28°C for 5 days.
Analytical determinations
Glucose, fructose, ethanol, glycerol, acetic acid, acetaldehyde, and malic acid were analyzed with enzymatic test kits using standard solution as assay control (R-Biopharm AG, Darmstadt, Germany). Titratable acidity was measured by acid–base titration with standardized 0.1 M NaOH.
The analysis of main esters related to wine aroma was conducted using gas chromatography (GC). The extraction of volatile compounds from the wine samples was done by addition of 5 ml diethyl ether to 3 ml of each wine, followed by vigorous shaking for about 2 min, and centrifugation (1,300g for 5 min). The top solvent layer was then transferred to a vial [29].
A gas chromatograph (Agilent 6890N, Agilent Technologies, CA, USA) equipped with a flame ionization detector (FID) was used. An HP-5 column (length 30 m, i.d. 0.32 mm, thickness 0.25 μm) (Hewlett-Packard, CA, USA) was utilized to analyze the following esters: ethyl acetate, ethyl caproate, ethyl caprylate, isoamyl acetate, 2-phenylethyl acetate. The operating conditions were as follows: the oven temperature programmed at 30°C for 5 min, followed by increasing the temperature to 250°C at 10°C/min and held at 250°C for 2 min. Sample injections of 1 μl were performed in splitless mode. The injector and FID detector temperatures were 270 and 300°C, respectively. Column flow rate was 1 ml/min using nitrogen as carrier gas. The FID output signal was recorded and processed using appropriate software (Agilent ChemStation Software, Agilent, CA, USA). Esters were quantified from the regression curve (R 2 > 98%) of the corresponding standard (Sigma, MO, USA), using external standard calibration and the GC conditions described above. In order to check the recovery efficiency of each ester from the wine sample, a known concentration of standard solution was added to each sample. Correction factors were calculated and used to adjust ester concentration values accordingly as described by Abeijón Mukdsi et al. [1].
The color of different wines was measured on samples diluted 1:10 with buffer (0.1 M citric acid with 0.2 M Na2HPO4, pH 4.1). The absorbance (A) of the samples was read at 420, 520, and 700 nm. The color intensity and hue were calculated as described by Wrolstad [51] using the equations [(A 520 – A 700) + (A 420 – A 700)] and [(A 520 – A 700)/(A 420 – A 700)], respectively.
Sensory analysis
Sensory descriptive analysis of the young wines (1 month after bottling) was carried out by a tasting panel that consisted of six trained judges (Bianchi Winery and National Institute of Viticulture, Mendoza, Argentina). Wines were equilibrated at room temperature (22°C) and 50 ml-samples were poured into randomly numbered wineglasses. Two consecutive sessions were performed on different days. The selection of sensory descriptors was done by the panelists during the first session, taking into account those that allow discrimination among treatments [18]. At the second session the intensity of each descriptor was rated on a scale from 0 (not perceivable) to 5 (very strong). The violet color, astringency, bitterness, floral aroma, fruity aroma, phenolic aroma, and equilibrium-harmony were tested.
Statistical analysis
Experiments were performed in duplicate. One-way analysis of variance (ANOVA) was applied to the experimental data and the Tukey test was used for multiple mean comparisons. All statistical analyses were performed with Statistica software version 7.
Results and discussion
Evolution of yeast microbial populations and fermentation kinetics
Because most of the non-Saccharomyces wine-related species show limited fermentation aptitudes, they may be only employed as starter cultures in conjunction with S. cerevisiae strains, in this way ensuring the completion of the AF [6, 34, 41].
Viable cell counts during microfermentations of non-Saccharomyces (filled diamonds, open diamonds) and Saccharomyces yeasts (filled squares, open squares) in pure and mixed cultures, respectively
Kinetics of alcoholic fermentations carried out by pure K. apiculata mc1 culture (filled diamonds), pure S. cerevisiae mc2 culture (filled squares), mixed K. apiculata/S. cerevisiae culture (open triangles) in microvinifications conditions
Influence of O. oeni X2L on microbial populations and malic acid evolution
Most red and some white wines undergo MLF, which is often encouraged during the final phases of AF or after its conclusion to avoid stuck AF by LAB strains or increased concentrations of acetic acid that render the wines unacceptable for consumption [7, 8]. However, differences between sequential and simultaneous AF and MLF are still controversial, specifically with regard to the effect on wine composition.
Malic acid consumption (filled triangles) and evolution of cell population of K. apiculata mc1 (open diamonds), S. cerevisiae mc2 (open squares), and O. oeni X2L (filled circles) in simultaneous (a) and sequential (b) inoculations
Sequential inoculation of O. oeni after completion of the AF led to a decrease in bacterial cell population and growth rate compared with microvinifications with simultaneous inoculation (Fig. 3b). However, after 5 days of incubation complete depletion of malic acid was observed.
The influence of the time of O. oeni inoculation on malolactic activity seems to be strain-specific for yeasts and bacteria used in wine fermentations. Jussier et al. [21] reported that simultaneous inoculation of yeasts and bacteria led to faster malic acid degradation, whereas Massera et al. [30] found that utilization of malic acid was similar for both inoculation strategies.
Chemical and sensorial analysis of young wines
Table 1 shows the chemical characteristics of wines fermented by different starter cultures. Sugars in microvinifications conducted by S. cerevisiae in pure or mixed cultures were completely consumed and the dryness of the must was achieved at the end of the AF. Ethanol concentrations reached standard values (12–13%). Similar results have been observed by Rodríguez et al. [41] who found that wines obtained with pure and mixed cultures of S. cerevisiae MMf9 showed physical–chemical characteristics like most regular wines. However, products fermented by single cultures of K. apiculata showed high residual sugar and low ethanol contents. The stuck fermentation was due to the growth kinetics exhibited by this apiculate yeast. Rodríguez et al. [41] indicated that the general composition of wine obtained with a single culture of C. pulcherrima V6 was consistent with its stuck fermentation kinetics.
General characteristics of wines fermented by different starter cultures
| Analytical determinations* | Pure starter cultures | Composite starter cultures | |||
|---|---|---|---|---|---|
| K | S | K + S | K + S + O (simultaneous) | K + S + O (sequential) | |
| Residual sugars (g/l) | 20.84 ± 0.97a | 0.72 ± 0.08b | 0.81 ± 0.06b | 0.86 ± 0.04b | 0.75 ± 0.05b |
| Ethanol (% v/v) | 8.82 ± 0.56a | 13.91 ± 0.27b | 13.56 ± 0.19b | 12.97 ± 0.44c | 13.38 ± 0.29bc |
| Glycerol (g/l) | 7.98 ± 0.23a | 8.55 ± 0.31b | 8.43 ± 0.22b | 8.27 ± 0.24ab | 8.16 ± 0.30ab |
| Volatile acidity (g/l) | 0.79 ± 0.04a | 0.45 ± 0.03b | 0.58 ± 0.03c | 1.23 ± 0.05d | 0.61 ± 0.02c |
| Acetaldehyde (mg/l) | 37.83 ± 1.37a | 56.54 ± 2.32b | 52.35 ± 1.95b | 17.76 ± 1.13c | 48.14 ± 2.11d |
| Titratable acidity (g/l) | 5.27 ± 0.20a | 5.91 ± 0.34b | 6.22 ± 0.32b | 6.79 ± 0.21c | 4.90 ± 0.37d |
| l-Malic acid (g/l) | 1.89 ± 0.03a | 1.76 ± 0.02b | 1.81 ± 0.03b | 0.04 ± 0.01c | 0.02 ± 0.01c |
| pH | 3.81 ± 0.02a | 3.83 ± 0.02a | 3.81 ± 0.02a | 3.92 ± 0.03b | 4.05 ± 0.03c |
| Color intensity | 1.60 ± 0.006a | 1.62 ± 0.036a | 1.59 ± 0.028a | 1.52 ± 0.009b | 1.70 ± 0.018c |
| Color hue | 1.24 ± 0.008a | 1.19 ± 0.003b | 1.22 ± 0.001c | 1.20 ± 0.004b | 1.15 ± 0.001d |
| Analytical determinations* | Pure starter cultures | Composite starter cultures | |||
|---|---|---|---|---|---|
| K | S | K + S | K + S + O (simultaneous) | K + S + O (sequential) | |
| Residual sugars (g/l) | 20.84 ± 0.97a | 0.72 ± 0.08b | 0.81 ± 0.06b | 0.86 ± 0.04b | 0.75 ± 0.05b |
| Ethanol (% v/v) | 8.82 ± 0.56a | 13.91 ± 0.27b | 13.56 ± 0.19b | 12.97 ± 0.44c | 13.38 ± 0.29bc |
| Glycerol (g/l) | 7.98 ± 0.23a | 8.55 ± 0.31b | 8.43 ± 0.22b | 8.27 ± 0.24ab | 8.16 ± 0.30ab |
| Volatile acidity (g/l) | 0.79 ± 0.04a | 0.45 ± 0.03b | 0.58 ± 0.03c | 1.23 ± 0.05d | 0.61 ± 0.02c |
| Acetaldehyde (mg/l) | 37.83 ± 1.37a | 56.54 ± 2.32b | 52.35 ± 1.95b | 17.76 ± 1.13c | 48.14 ± 2.11d |
| Titratable acidity (g/l) | 5.27 ± 0.20a | 5.91 ± 0.34b | 6.22 ± 0.32b | 6.79 ± 0.21c | 4.90 ± 0.37d |
| l-Malic acid (g/l) | 1.89 ± 0.03a | 1.76 ± 0.02b | 1.81 ± 0.03b | 0.04 ± 0.01c | 0.02 ± 0.01c |
| pH | 3.81 ± 0.02a | 3.83 ± 0.02a | 3.81 ± 0.02a | 3.92 ± 0.03b | 4.05 ± 0.03c |
| Color intensity | 1.60 ± 0.006a | 1.62 ± 0.036a | 1.59 ± 0.028a | 1.52 ± 0.009b | 1.70 ± 0.018c |
| Color hue | 1.24 ± 0.008a | 1.19 ± 0.003b | 1.22 ± 0.001c | 1.20 ± 0.004b | 1.15 ± 0.001d |
Mean values with different superscript letters within the same row are significantly different according to the Tukey test (P ≤ 0.05)
Data are mean values of two experiments ± standard deviation
K, K. apiculata mc1; S, S. cerevisiae mc2; O, O. oeni X2L
*Evaluated in the finished wines
General characteristics of wines fermented by different starter cultures
| Analytical determinations* | Pure starter cultures | Composite starter cultures | |||
|---|---|---|---|---|---|
| K | S | K + S | K + S + O (simultaneous) | K + S + O (sequential) | |
| Residual sugars (g/l) | 20.84 ± 0.97a | 0.72 ± 0.08b | 0.81 ± 0.06b | 0.86 ± 0.04b | 0.75 ± 0.05b |
| Ethanol (% v/v) | 8.82 ± 0.56a | 13.91 ± 0.27b | 13.56 ± 0.19b | 12.97 ± 0.44c | 13.38 ± 0.29bc |
| Glycerol (g/l) | 7.98 ± 0.23a | 8.55 ± 0.31b | 8.43 ± 0.22b | 8.27 ± 0.24ab | 8.16 ± 0.30ab |
| Volatile acidity (g/l) | 0.79 ± 0.04a | 0.45 ± 0.03b | 0.58 ± 0.03c | 1.23 ± 0.05d | 0.61 ± 0.02c |
| Acetaldehyde (mg/l) | 37.83 ± 1.37a | 56.54 ± 2.32b | 52.35 ± 1.95b | 17.76 ± 1.13c | 48.14 ± 2.11d |
| Titratable acidity (g/l) | 5.27 ± 0.20a | 5.91 ± 0.34b | 6.22 ± 0.32b | 6.79 ± 0.21c | 4.90 ± 0.37d |
| l-Malic acid (g/l) | 1.89 ± 0.03a | 1.76 ± 0.02b | 1.81 ± 0.03b | 0.04 ± 0.01c | 0.02 ± 0.01c |
| pH | 3.81 ± 0.02a | 3.83 ± 0.02a | 3.81 ± 0.02a | 3.92 ± 0.03b | 4.05 ± 0.03c |
| Color intensity | 1.60 ± 0.006a | 1.62 ± 0.036a | 1.59 ± 0.028a | 1.52 ± 0.009b | 1.70 ± 0.018c |
| Color hue | 1.24 ± 0.008a | 1.19 ± 0.003b | 1.22 ± 0.001c | 1.20 ± 0.004b | 1.15 ± 0.001d |
| Analytical determinations* | Pure starter cultures | Composite starter cultures | |||
|---|---|---|---|---|---|
| K | S | K + S | K + S + O (simultaneous) | K + S + O (sequential) | |
| Residual sugars (g/l) | 20.84 ± 0.97a | 0.72 ± 0.08b | 0.81 ± 0.06b | 0.86 ± 0.04b | 0.75 ± 0.05b |
| Ethanol (% v/v) | 8.82 ± 0.56a | 13.91 ± 0.27b | 13.56 ± 0.19b | 12.97 ± 0.44c | 13.38 ± 0.29bc |
| Glycerol (g/l) | 7.98 ± 0.23a | 8.55 ± 0.31b | 8.43 ± 0.22b | 8.27 ± 0.24ab | 8.16 ± 0.30ab |
| Volatile acidity (g/l) | 0.79 ± 0.04a | 0.45 ± 0.03b | 0.58 ± 0.03c | 1.23 ± 0.05d | 0.61 ± 0.02c |
| Acetaldehyde (mg/l) | 37.83 ± 1.37a | 56.54 ± 2.32b | 52.35 ± 1.95b | 17.76 ± 1.13c | 48.14 ± 2.11d |
| Titratable acidity (g/l) | 5.27 ± 0.20a | 5.91 ± 0.34b | 6.22 ± 0.32b | 6.79 ± 0.21c | 4.90 ± 0.37d |
| l-Malic acid (g/l) | 1.89 ± 0.03a | 1.76 ± 0.02b | 1.81 ± 0.03b | 0.04 ± 0.01c | 0.02 ± 0.01c |
| pH | 3.81 ± 0.02a | 3.83 ± 0.02a | 3.81 ± 0.02a | 3.92 ± 0.03b | 4.05 ± 0.03c |
| Color intensity | 1.60 ± 0.006a | 1.62 ± 0.036a | 1.59 ± 0.028a | 1.52 ± 0.009b | 1.70 ± 0.018c |
| Color hue | 1.24 ± 0.008a | 1.19 ± 0.003b | 1.22 ± 0.001c | 1.20 ± 0.004b | 1.15 ± 0.001d |
Mean values with different superscript letters within the same row are significantly different according to the Tukey test (P ≤ 0.05)
Data are mean values of two experiments ± standard deviation
K, K. apiculata mc1; S, S. cerevisiae mc2; O, O. oeni X2L
*Evaluated in the finished wines
Glycerol contents were similar in all products (8–8.5 g/l) and these values are in accordance with those usually found in wines, between 5 and 20 g/l [40].
Acetic acid becomes unpleasant at concentrations near its sensory threshold (0.7–1.0 g/l) and values between 0.2 and 0.7 g/l are usually considered adequate [25]. Levels of volatile acidity were significantly higher in fermentations with pure cultures of the non-Saccharomyces yeast or mixed starter cultures of yeasts and bacterium inoculated simultaneously. Under the latter conditions, the acetic acid concentration in the fermented product was the highest (1.23 g/l) and could affect the wine organoleptic characteristics. It has been reported that at elevated sugar concentrations this mostly heterofermentative wine LAB can produce high acetic acid concentrations through its sugar metabolism [24, 28]. However, other authors claim that simultaneous fermentations did not affect acetic acid levels [22, 46].
Acetaldehyde is another important flavor-active fermentation compound with average values of about 40 and 80 mg/l for red and white wines, respectively [26]. Acetaldehyde concentrations in the different wines assayed depended on the starter culture utilized in the microfermentation. An important decrease in this metabolite was observed in wines with simultaneous fermentation of yeasts and O. oeni. This would indicate that the bacterium was able to metabolize acetaldehyde under the given assay conditions, which is in agreement with previous studies that showed that simultaneous fermentations displayed the lowest overall acetaldehyde concentrations during the AF, likely because of the degradation of this compound by bacteria [21]. Furthermore, Osborne et al. [37] reported degradation of acetaldehyde by homofermentative and heterofermentative LAB and the two major catabolic products were identified as ethanol and acetic acid. However, those authors believe that the impact of both products on the chemical and sensory composition of wine is limited, because the increase in ethanol and acetic acid from acetaldehyde degradation would be insignificant. Wines obtained with simultaneous inoculation of yeasts and O. oeni showed an increase in acetic acid and this behavior could in part be due to acetaldehyde degradation (Table 1). Other studies found that LAB also degraded citric acid with acetic acid as the end product [21, 36].
Wines with MLF induction after the AF showed a titratable acidity significantly lower than in other products fermented by mixed starter cultures. In addition, inoculation of O. oeni increased the wine pH because of bacterial consumption of l-malic acid.
Fermentation-derived esters are largely responsible for wine fruitiness, and therefore they play an important role in the sensory composition of young red and white wines [10, 33]. The final concentration of esters compounds in wine is the result of the balance between alcohol acetyltransferase enzymes promoting their synthesis, and esterase enzymes promoting their hydrolysis [13, 39]. The two main groups of esters that have long been associated with wine fruitiness are acetate and ethyl esters that are recognized as important flavor compounds [42, 43]. Ethyl acetate, the major ester in wine can produce a solvent-like odor at concentrations beyond the threshold of 150–200 mg/l [25], whereas at levels of 80 mg/l it contributes to fruity notes and adds to a general complexity [15, 19]. Despite the high production of ethyl acetate in single cultures of K. apiculata mc1, in trials conducted using mixed culture a reduction of this ester was observed (Table 2).
Esters concentrations of wines fermented by pure and mixed starter cultures
| Pure starter cultures | Composite starter cultures | ||||
|---|---|---|---|---|---|
| K | S | K + S | K + S + O (simultaneous) | K + S + O (sequential) | |
| Ethyl acetate (mg/l) | 178.84 ± 5.67a | 29.21 ± 0.92b | 79.73 ± 1.31c | 76.92 ± 1.63c | 68.33 ± 1.21d |
| Ethyl caproate (mg/l) | 0.65 ± 0.06a | 1.23 ± 0.08b | 1.31 ± 0.04c | 1.12 ± 0.05b | 1.41 ± 0.07d |
| Ethyl caprylate (mg/l) | 0.41 ± 0.05a | 0.92 ± 0.04b | 1.12 ± 0.03c | 0.83 ± 0.06b | 1.02 ± 0.05c |
| Isoamyl acetate (mg/l) | 3.73 ± 0.22a | 0.94 ± 0.06b | 2.32 ± 0.13c | 2.81 ± 0.15d | 2.13 ± 0.11c |
| 2-Phenylethyl acetate (mg/l) | 29.42 ± 1.21a | 0.94 ± 0.03b | 12.81 ± 0.93c | 9.95 ± 0.52d | 11.51 ± 0.73c |
| Pure starter cultures | Composite starter cultures | ||||
|---|---|---|---|---|---|
| K | S | K + S | K + S + O (simultaneous) | K + S + O (sequential) | |
| Ethyl acetate (mg/l) | 178.84 ± 5.67a | 29.21 ± 0.92b | 79.73 ± 1.31c | 76.92 ± 1.63c | 68.33 ± 1.21d |
| Ethyl caproate (mg/l) | 0.65 ± 0.06a | 1.23 ± 0.08b | 1.31 ± 0.04c | 1.12 ± 0.05b | 1.41 ± 0.07d |
| Ethyl caprylate (mg/l) | 0.41 ± 0.05a | 0.92 ± 0.04b | 1.12 ± 0.03c | 0.83 ± 0.06b | 1.02 ± 0.05c |
| Isoamyl acetate (mg/l) | 3.73 ± 0.22a | 0.94 ± 0.06b | 2.32 ± 0.13c | 2.81 ± 0.15d | 2.13 ± 0.11c |
| 2-Phenylethyl acetate (mg/l) | 29.42 ± 1.21a | 0.94 ± 0.03b | 12.81 ± 0.93c | 9.95 ± 0.52d | 11.51 ± 0.73c |
Mean values with different superscript letters within the same row are significantly different according to the Tukey test (P ≤ 0.05)
Data are mean values of two experiments ± standard deviation
K, K. apiculata mc1; S, S. cerevisiae mc2; O, O. oeni X2L
Esters concentrations of wines fermented by pure and mixed starter cultures
| Pure starter cultures | Composite starter cultures | ||||
|---|---|---|---|---|---|
| K | S | K + S | K + S + O (simultaneous) | K + S + O (sequential) | |
| Ethyl acetate (mg/l) | 178.84 ± 5.67a | 29.21 ± 0.92b | 79.73 ± 1.31c | 76.92 ± 1.63c | 68.33 ± 1.21d |
| Ethyl caproate (mg/l) | 0.65 ± 0.06a | 1.23 ± 0.08b | 1.31 ± 0.04c | 1.12 ± 0.05b | 1.41 ± 0.07d |
| Ethyl caprylate (mg/l) | 0.41 ± 0.05a | 0.92 ± 0.04b | 1.12 ± 0.03c | 0.83 ± 0.06b | 1.02 ± 0.05c |
| Isoamyl acetate (mg/l) | 3.73 ± 0.22a | 0.94 ± 0.06b | 2.32 ± 0.13c | 2.81 ± 0.15d | 2.13 ± 0.11c |
| 2-Phenylethyl acetate (mg/l) | 29.42 ± 1.21a | 0.94 ± 0.03b | 12.81 ± 0.93c | 9.95 ± 0.52d | 11.51 ± 0.73c |
| Pure starter cultures | Composite starter cultures | ||||
|---|---|---|---|---|---|
| K | S | K + S | K + S + O (simultaneous) | K + S + O (sequential) | |
| Ethyl acetate (mg/l) | 178.84 ± 5.67a | 29.21 ± 0.92b | 79.73 ± 1.31c | 76.92 ± 1.63c | 68.33 ± 1.21d |
| Ethyl caproate (mg/l) | 0.65 ± 0.06a | 1.23 ± 0.08b | 1.31 ± 0.04c | 1.12 ± 0.05b | 1.41 ± 0.07d |
| Ethyl caprylate (mg/l) | 0.41 ± 0.05a | 0.92 ± 0.04b | 1.12 ± 0.03c | 0.83 ± 0.06b | 1.02 ± 0.05c |
| Isoamyl acetate (mg/l) | 3.73 ± 0.22a | 0.94 ± 0.06b | 2.32 ± 0.13c | 2.81 ± 0.15d | 2.13 ± 0.11c |
| 2-Phenylethyl acetate (mg/l) | 29.42 ± 1.21a | 0.94 ± 0.03b | 12.81 ± 0.93c | 9.95 ± 0.52d | 11.51 ± 0.73c |
Mean values with different superscript letters within the same row are significantly different according to the Tukey test (P ≤ 0.05)
Data are mean values of two experiments ± standard deviation
K, K. apiculata mc1; S, S. cerevisiae mc2; O, O. oeni X2L
The next highest ester concentration corresponded to 2-phenylethyl acetate, which contributes to fruity and flowery notes [25]. Our results demonstrate that products fermented by composite cultures of K. apiculata and S. cerevisiae showed statistically higher concentrations of acetate esters such as isoamyl acetate and 2-phenylethyl acetate compared to wines fermented with S. cerevisiae monocultures (Table 2). This characteristic could be due to inoculation with the non-Saccharomyces strain, showing that this yeast could improve the wine aroma. Previous studies have already reported that non-Saccharomyces yeasts are good producers of several esters. Hanseniaspora/Kloeckera species were shown to be strong producers of ethyl acetate and 2-phenylethyl acetate [43, 49, 50].
With respect to ethyl esters the genus Saccharomyces is the best producer of ethyl caprylate and ethyl caproate [25, 43]. Our assays revealed that ethyl ester levels in mixed fermentations were similar to those produced by a pure culture of S. cerevisiae. Positive interactions between wild yeasts and S. cerevisiae cultures regarding volatile aroma compounds have been described by several authors who observed an increase in ester concentrations in wines produced by composite starters in comparison with pure fermentations [14, 34, 48].
All products fermented with a mixture of yeasts and O. oeni, independently of the timing of bacterial inoculation, showed a similar composition of the analyzed aroma compounds and only a slight decrease in the ester concentrations was observed. Matthews et al. [31] showed that LAB possess esterase activities that could potentially alter the ester profile of wine. Also the authors reported that O. oeni has higher esterase activity than lactobacilli and pediococci.
Cobweb graph of scores obtained from sensory analysis for wines fermented by pure K. apiculata mc1 culture (filled diamonds), pure S. cerevisiae mc2 culture (filled squares), mixed K. apiculata/S. cerevisiae culture (open triangles), and composite culture of yeasts and malobacterium with simultaneous (open circles) and sequential (filled circles) inoculations
Products simultaneously fermented by yeasts and O. oeni scored the highest for phenolic aroma and consequently obtained the lowest equilibrium-harmony ratings (Fig. 4). This is contrary to wines sequentially inoculated with malolactic bacterium, which had the highest acceptance, with better fruity and floral aromas and high scores for the equilibrium-harmony descriptor. These results are in disagreement with those reported by Massera et al. [30] who, working with Malbec must from another region of Argentina, found that wines with simultaneous treatment showed enhanced sensorial attributes related to high-quality wine like color and fruity flavor. Perhaps, this behavior was correlated with the different fermentation kinetics of the microorganisms employed in both studies. Whereas in our fermentation trials, O. oeni X2L represents an autochthonous microorganism from the northwest region of Argentina, Massera et al. [30] used a freeze-dried commercial O. oeni strain to inoculate regional musts.
In conclusion, red wines obtained with simultaneous inoculation of malolactic bacterium, non-Saccharomyces, and Saccharomyces yeasts were not accepted by our judges because of their strong phenolic aroma and high level of volatile acidity. However, wines fermented by mixed starter cultures of yeasts and sequential inoculation of O. oeni scored the highest for positive descriptors and allowed better control of the sensory quality of red wines from vineyards of Argentina.
The results of the present study allow us to propose the inclusion of K. apiculata mc1 as an adjunct culture to S. cerevisiae mc2 during must fermentation to improve the organoleptic properties of Malbec wines. In addition, sequential inoculation of O. oeni X2L after the AF enhanced sensory characteristics. Nevertheless, more specific information is required about the contribution of these strains. Large-scale experiments should be carried out to confirm the behavior of the mixed starter cultures proposed in this work.
To our knowledge, this is the first study on red wine obtained with indigenous strains of yeasts and bacterium from the northwest of Argentina, a region with growing oenological relevance.
Acknowledgments
This work was financially supported by Consejo Nacional de Investigaciones Científicas y Técnicas and Consejo de Investigaciones de la Universidad Nacional de Tucumán, Argentina. The authors are grateful to Elena Bru for her assistance in statistical analysis.
References
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