Effect of Aging on Lees on the Volatile Profile of Malvasia aromatica Wines Fermented with Saccharomyces Native Yeasts in PDO “Vinos de Madrid”

Abstract

The loss of aromatic and sensory quality in wines because of climate change in traditional winemaking areas is a challenge for winemakers. Aging on lees of the wine fermented with Saccharomyces native yeasts has been tested as a technique to try to improve the sensory characteristics of Malvasia aromatica white wines in PDO “Vinos de Madrid.” The grapes were pre-cold macerated and fermented with S. cerevisiae CLI 271 and CLI 889 (native yeast strains). Then, commercial lees were used for aging of wines for a five-month at low temperature in order to compare with the effect of S. cerevisiae CLI 271 and CLI 889 without lees treatment. Aromatic and organoleptic properties of wines aged on lees were studied using GC-FID and HS-SPME/GC–MS to quantify volatile compounds and a taster panel to sensorial analysis. There was a significant decrease in the ester family in wines aged on lees being more pronounced in CLI 889 wines. The treatment contributed to enhance the fruity and floral aromatic properties in CLI 271 and CLI 889 wines, respectively according to tasting panel, which showed a hedonic preference for CLI 271 wines without lees treatment and CLI 889 wines aged on lees.

1. Introduction

Aging on lees is a technique used after fermentation associated with the improvement of the sensory properties of wine through yeast autolysis via enzymatic self-degradation of cellular constituents that begins after the death of the yeast. It is a technique used for decades mainly in the production of white wines. Its main objective is to reduce astringency and bitterness, increase body, structure and roundness in the mouth and improve aromatic persistence and complexity of wines [1, 2, 3] in addition to contributing to the reduction of undesirable flavors [4]. The main conditioning and essential factor to allow cell autolysis is the time. It is estimated that the process begins as soon as the cell dies and increases significantly after the second month. The autolysis process is slow and involves risks of microbiological spoilage, reduction and organoleptic off-flavors [1, 5]. Therefore, aging on lees is combined with battonnages to improve the contact of lees with the wine and help the faster release of polysaccharides from the yeasts [6] and compounds such as amino acids and lipids that can be aroma precursors.

Aroma is one of the main quality attributes of white wines and a very important aspect for consumers. The volatile compounds provided by the grapes, more recognized as varietal aromas, are responsible for their aromatic typicity and their presence is influenced by factors such as variety, cultural practices, terroir, geographical location and climate [7]. The compounds released in the course of alcoholic fermentation also have a decisive influence on the volatile composition of wines and their synthesis depends mainly on the microorganisms involved.

PDO “Vinos de Madrid” is located in the center of the Iberian Peninsula with a prevalence of hot summers, cold winters and low levels of precipitation. Climate predictions indicate a gradual increase in temperatures, a decrease in precipitation and a greater frequency of severe phenomena such as frosts, storms and heat waves with greater incidence in the center of Spain [8]. These events could compromise the correct development of the technological and aromatic ripening of the grapes, preventing the production of intense aromatic white wines. On the other hand, the loss of aromatic and sensory typicity attached to a particular region is also a challenge for winemakers. The yeast activity during a spontaneous fermentation could contribute to fewer desirable attributes to the wine and the quality can be variable between seasons. Thus, the use of commercial yeasts could be employed to obtain a product of uniform quality [9, 10]. However, the typical character of the harvest as well as its aromatic properties could be lost.

In order to adapt to the climate change-related effects, as well as the preservation of the typical organoleptic characteristics of the Madrid region, our laboratories have proposed the use of oenological practices such as skin contact treatment and the use of indigenous yeast strains better adapted to climatic conditions and terroir [11, 12]. As a final step, based on the results of our previous research, this study has focused on the effect of using lees during the aging period in white wines of Malvasia aromatica in bottles. This variety was selected for having a great aromatic potential, becoming an interesting alternative in the use of new varieties in addition to the traditional ones that could help to improve the value of the local wines. The objective of this research was to optimize the combination of aging on lees techniques and the selection of the adequate native S. cerevisiae yeast strain for Vitis vinifera L.cv. Malvasia aromatica (Figure 1) winemaking in PDO “Vinos de Madrid”.

Figure 1.

Vitis vinifera L.cv. Malvasia aromatica [13].

2. Material and methods

2.1 Vintage, yeast strains and fermentation procedure

Grapes from the white-berried cv. Malvasia aromatica were collected and processed in the Experimental Winery from the IMIDRA Research Institute (Finca El Encín, Alcalá de Henares, Madrid, Spain). The yeast strains of Saccharomyces cerevisiae selected for this research were CLI 271 and CLI 889 used in this work were previously isolated from wineries of the PDO “Vinos de Madrid”, and selected due to their good fermentative and oenological performance [14, 15, 16]. Grape must be macerated at 10°C for 18 h before yeast inoculation at a concentration of 10⁶ cells/mL [11]. Fermentations took place in triplicate under a controlled temperature of 16°C and were monitored on a daily basis by measuring density.

2.2 Aging on lees

After the end of the alcoholic fermentation, samples of each wine were racked into 5 L screw-capped glass bottles. Commercial lees (Super Bouquet MN from Agrovin) were used at a concentration of 30 g/hl for a five-month contact period with wine lees (L) at low temperature (8–11°C). Five liters of each wine (without lees) were used as a control. Battonnages in wines aging on lees were carried out twice per week, for 10 minutes per bottle with the aid of a magnetic stirrer. At the end of this treatment, wines were racked to eliminate lees. Wines were also clarified and bottled in the same way as those without lees. Prior to bottling, samples were taken for analysis.

2.3 Wine volatile composition

Wine volatile composition was defined with: major aroma compounds and free varietal minor volatiles. The first were analyzed using gas chromatography coupled with a flame ionization detector (GC-FID). The second, by undertaking a headspace-solid phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME/GC–MS).

The analysis of major aromatic compounds included the extraction and detection of alcohols, acids, lactones, esters, aldehydes and ketones, according to the method described by Ortega [17], using dichloromethane and a DB-Wax column (60 m × 0.32 mm × 0.5 μm film thickness) from J&W Scientific (Folsom, CA, USA). For sample preparation, conical bottom glass tubes were used, adding 3.9 g of ammonium sulfate, 6.3 mL of milli-Q grade deionized water, 2.7 mL of wine, 20 μL of an internal standard solution (2-Butanol, 4-Methyl-2-pentanol, 4-hydroxy-4-methyl-2-pentanone and 2-Octanol) and 250 μL of dichloromethane. Chromatography conditions included an oven temperature initially programmed at 40°C for 5 min, and then ramped to 200°C. A constant helium flow of 2 mL/min was used. Two mL of aroma extract were injected at 250°C in splitless mode. The total run time was 75 min per sample.

Minor volatiles (terpenoids and C13-norisoprenoids) were determined using headspace-solid phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME/GC–MS), following the method proposed by Yuan and Qian [18] on an Agilent 6890 gas chromatograph equipped with an Agilent 5973 mass selective detector (Agilent). A volume of two mL of the wine sample was diluted with 8 mL of citric acid (0.5 g/L citric acid, pH 3 saturated with sodium chloride) and 20 μL of 4-octanol (100 μg/L), used as internal standard. The extraction was done by stirring the sample in vials for chromatography (20 mL of volume, Agilent Technologies) tightly capped with a magnetic stir bar for 10 min at 50°C in a thermostatic bath and using SPME fiber (50/30 μm DVB/CAR/PDMS fiber from Supelco Inc., Bellefonte, PA, USA) for 50 min at the same temperature with stirring (1000 rpm) to capture the volatiles. The fiber was manually inserted into the injection port of the GC at 230°C to desorb the analytes. A DB-Wax column from J&W Scientific (Folsom, CA, USA) (60 m × 0.32 mm × 0.5 μm film thickness, Phenomenex, Torrance, CA, USA) was employed to separate the analytes. Carrier gas (helium) was set at a constant flow rate of 1 mL/min. The oven temperature was initially set at 40°C for 2 min, and raised to 230°C at 5°C per min for 15 min. All samples were analyzed in duplicate.

2.4 Incidence of aroma composition

To estimate the contribution of volatile compounds to wine aroma, the odor activity value (OAV) was calculated by estimating the ratio between the concentration of each compound and its perception threshold. A compound was considered to contribute to wine aroma if OAV ≥ 1. The perception threshold used in this work were those found in the literature [19, 20, 21, 22, 23, 24, 25].

2.5 Sensorial analysis

Wines were tasted at the tasting room of Experimental Winery from IMIDRA Institute by a sensory panel of eight trained evaluators. Descriptive sensory analyses and triangle tests were performed following the indications of the ISO 4120:2004 to assess the effect of experimental treatments (use of lees) on wine aroma. Sensory descriptive analysis was performed to describe and quantify wine attributes from 1 (low intensity) to 10 (high intensity). The evaluation included a visual phase, an olfactory phase and a gustatory phase, which was interpreted by graphical representation. A hedonic classification was also carried out to determine the order of preference of the wines. A final score for each wine was obtained as the mean with their respective standard deviation.

2.6 Statistical analysis

Statistical analyses were performed using SPSS ver. 20.0 (SPSS, Inc., Chicago, IL, USA). Analysis of variance (ANOVA) was made to examine the differences between treatments in terms of volatile compounds and sensory attributes of the wines. In order to assess the significance (p < 0.05) of differences between means, Tukey honestly significant difference (HSD) post-hoc tests were used to establish the significance of differences between means to assess significance (p < 0.05).

3. Results and discussion

3.1 Wine volatile composition of Malvasia aromatica elaborated wines

An analysis of the aroma profile of Malvasia wines elaborated with the indigenous yeast strains CLI 271 and CLI 889 was made in both conditions, with and without aging on lees, to determine the effects of aging on lees on the volatile composition. Figure 2 and Table 1 show the evolution of volatile compounds in wines from the two yeasts after treatment on lees, grouped into chemical families.

Figure 2.

Effect of aging on lees on the levels of aromatic compounds grouped by families in Malvasia aromatica wines fermented with S. cerevisiae CLI 271 and CLI 889.

Compounds	OTH	CLI 271			CLI 271 L			Siga	CLI 889			CLI 889 L			Sig.a
Terpenols (μg l^−1)
β-Myrcene	—	1.17	±	0.22	1.55	±	0.06	*	1.86	±	0.06	2.17	±	0.27	Ns
α-Terpinene	—	0.22	±	0.03	0.22	±	0.06	Ns	0.17	±	0.06	0.26	±	0.02	Ns
Limonene	15b	0.53	±	0.08	0.56	±	0.08	Ns	0.49	±	0.08	0.79	±	0.08	*
γ-Terpinene	—	1.23	±	0.12	1.83	±	0.10	**	1.89	±	0.10	2.49	±	0.27	*
Linalool	25c	69.89	±	5.52	75.14	±	8.29	Ns	78.55	±	8.29	84.17	±	4.79	Ns
α-Terpineol	250c	25.21	±	4.12	25.26	±	2.06	Ns	23.39	±	2.06	23.86	±	1.31	Ns
β-Citronellol	100d	12.07	±	1.12	11.43	±	2.05	Ns	17.31	±	2.05	16.99	±	0.96	Ns
Geraniol	30b	19.17	±	2.78	16.65	±	3.01	Ns	20.74	±	3.01	19.69	±	1.53	Ns
Total		129.48	±	13.97	132.63	±	15.71		144.40	±	15.71	150.41	±	9.22
C13-norisoprenoids (μg l^−1)
β-Damascenone	0.05d	1.24	±	0.06	1.16	±	0.14	Ns	1.11	±	0.02	1.33	±	0.04	**
Total		1.24	±	0.06	1.16	±	0.14		1.11	±	0.02	1.33	±	0.04
Alcohols (mg l^−1)
Isobutanol	40c	35.58	±	1.74	28.19	±	1.08	**	42.30	±	0.44	37.70	±	0.17	***
1-Butanol	150b	1.03	±	0.05	0.90	±	0.05	*	0.40	±	0.02	0.35	±	0.03	Ns
Isoamyl alcohol	30c	296.05	±	27.63	262.31	±	22.26	Ns	315.63	±	16.13	309.26	±	16.92	Ns
1-Hexanol	8c	1.61	±	0.01	1.26	±	0.01	***	1.58	±	0.03	1.49	±	0.04	*
Methionol	1c	1.80	±	0.19	1.36	±	0.04	*	0.85	±	0.07	0.68	±	0.00	*
β-Phenylethanol	14c	56.33	±	3.21	44.91	±	1.86	*	34.30	±	2.20	31.11	±	0.53	Ns
Total		392.41	±	32.82	338.95	±	25.31		395.09	±	18.90	380.62	±	17.70
Lactones (mg l^−1)
γ -Butyrolactone	35e	6.33	±	0.98	3.53	±	0.28	**	7.35	±	1.25	6.34	±	1.27	Ns
Total		6.33	±	0.98	3.53	±	0.28		7.35	±	1.25	6.34	±	1.27
Fatty acids (mg l^−1)
Isobutyric acid	2.30f	1.39	±	0.21	1.00	±	0.11	*	1.72	±	0.11	1.39	±	0.10	*
Butyric acid	0.17c	0.64	±	0.08	0.41	±	0.02	**	0.73	±	0.11	0.65	±	0.11	Ns
Isovaleric acid	0.03c	2.43	±	0.30	1.79	±	0.18	*	2.09	±	0.13	1.67	±	0.07	**
Hexanoic acid	0.42c	2.92	±	0.62	3.03	±	0.22	Ns	2.31	±	0.17	2.44	±	0.11	Ns
Octanoic acid	0.50c	4.01	±	0.81	4.22	±	0.22	Ns	3.27	±	0.26	3.23	±	0.02	Ns
Decanoic acid	1c	0.41	±	0.12	0.37	±	0.00	Ns	0.34	±	0.02	0.30	±	0.01	*
Total		11.80	±	2.14	10.81	±	0.76		10.47	±	0.80	9.67	±	0.42
Esters (mg l^−1)
Ethyl butyrate	0.02c	0.27	±	0.01	0.20	±	0.01	**	0.31	±	0.02	0.28	±	0.01	Ns
Ethyl isovalerate	0.003c	0.26	±	0.04	0.17	±	0.01	*	0.26	±	0.01	0.22	±	0.03	Ns
Isoamyl acetate	0.03c	1.68	±	0.19	1.40	±	0.01	Ns	1.11	±	0.03	0.97	±	0.04	*
Ethyl hexanoate	0.01c	0.54	±	0.12	0.38	±	0.01	Ns	0.52	±	0.03	0.36	±	0.00	**
Hexyl acetate	1g	0.10	±	0.01	0.10	±	0.00	Ns	0.10	±	0.00	0.04	±	0.00	***
Ethyl lactate	154b	15.48	±	2.86	9.72	±	0.91	*	17.16	±	0.84	10.67	±	0.40	***
Ethyl octanoate	0.58b	0.52	±	0.08	0.34	±	0.00	*	0.51	±	0.03	0.26	±	0.01	***
Ethyl 3-Hydroxy-butyrate	20e	0.03	±	0.06	0.04	±	0.04	Ns	0.11	±	0.00	0.09	±	0.00	***
Diethyl succinate	1.20c	0.56	±	0.22	0.24	±	0.01	Ns	1.17	±	0.00	0.51	±	0.01	**
2-Phenylethyl acetate	0.25c	0.26	±	0.01	0.34	±	0.01	**	0.12	±	0.00	0.11	±	0.00	*
Total		19.71	±	3.62	12.93	±	1.00		21.38	±	0.98	13.51	±	0.49
Carbonyl comp. (mg l^−1)
Diacetyl	0.10d	0.16	±	0.05	0.11	±	0.01	Ns	0.23	±	0.02	0.11	±	0.02	**
Acetoin	150h	0.04	±	0.00	Tr			Ns	0.30	±	0.07	0.03	±	0.03	**
Benzaldehyde	5c	0.18	±	0.04	0.14	±	0.02	Ns	0.21	±	0.01	0.17	±	0.01	**
Total		0.38	±	0.08	0.25	±	0.03		0.44	±	0.03	0.28	±	0.03
Total (mg l^−1)		430.77	±	39.6	366.62	±	27.41		434.88	±	21.97	410.57	±	19.93

Table 1.

Effect of aging on lees (L) on the aroma compound levels of Malvasia aromatica wines fermented with S. cerevisiae CLI 271 and CLI 889.

^a

Significance at which means differ as shown by analysis of variance: * p < 0.05; ** p < 0.01; *** p < 0.001.

^b

[18]

^c

[19]

^d

[20]

^e

[21]

^f

[22]

^g

[23]

^h

[24].

OTH^*: Odor threshold value.

Ns: not significant.

Tr: traces.

In bold the compounds that seem to have a clear connection with aging on lees treatment.

Thirty-five volatiles were quantified, and distributed in varietal aromas (terpenols and C13-norisoprenoids), alcohols, acids, lactones, esters, aldehydes and ketones; their total content is given in μg/L for varietal aromas and mg/L for the rest of the compounds. Table 1 also shows the results of the analysis of variance (ANOVA) for each fermentation to determine the compounds that show significant individual differences due to the effect of aging on lees.

The effect of lees treatment was significant in 57% of the compounds identified in wines fermented with CLI 889 strain (20 of 35 compounds identified) and 45% (16 of 35 compounds) in wines from CLI 271 strain (Table 1).

Aging on lees caused a significant decrease in the total content of esters, aldehydes and carbonyl compounds in the two winemaking processes (Figure 2). The same behavior for the ester family was reported by Bueno et al. [26] and Del Barrio-Galán et al. [27] in Macabeo and Verdejo varieties respectively after a period of aging on lees.

The ANOVA analysis in Table 1 shows major differences in the CLI 889 wines, 8 of the 10 esters identified decreased significantly. Some studies have shown how the interactions between macromolecules released by the lees and aromatic compounds present in the wine could modify the volatility and aromatic intensity of these compounds; thus, the lower content of some esters compared to the control wine (without lees) could be explained by their association with mannoproteins and polysaccharides released, reducing volatility. These results are in agreement with other authors on model wines [28, 29]. On the other hand, in the esters where there was a notable decrease in terms of concentration, it was not decisive in terms of aroma contribution. Despite the loss, they continued to exceed their perception threshold (isoamyl acetate and ethyl hexanoate) or did not reach the threshold before lees treatment (hexyl acetate, ethyl lactate, ethyl octanoate, diethyl succinate and ethyl 3-hydroxy butyrate). Some of these compounds also showed lower concentrations in Tempranillo wines aged on lees [30]. The authors explained that the lower content of these volatiles can be due to an interaction between esters, acetates and mannoproteins from yeast autolysis and/or the sorption phenomenon onto yeast cell walls. This behavior was also denoted in model wines [28, 29, 31] and in white wines [26]. It could be happened in Malvasia wines from the present work. Wines fermented with CLI 271 showed a significant decrease in 4 of the 10 esters quantified. This decrease was not a disadvantage in terms of aroma contribution because ethyl butyrate and ethyl isovalerate still exceeded their perception threshold despite the loss of concentration and ethyl lactate and ethyl octanoate did not reach the threshold before lees treatment. It is important to note the role of 2-phenylethyl acetate in CLI 271 wines. Its concentration increases significantly with lees treatment. This compound is related to fruity attributes.

Regarding carbonyl compounds, the significant decrease of acetoin (creamy and buttery aromas) in CLI 889 L wines is noteworthy. Acetoin on its own does not have a significant aromatic impact due to its high detection threshold, although it does contribute to the wine bouquet due to its interrelation in the synthesis of diacetyl [32], a compound that, although it decreased significantly in CLI 889 wines, exceeds its perception threshold, contributing buttery aromas. Benzaldehyde also decreased significantly in CLI 889 wines but in all cases, like acetoin, it was far from its threshold.

The effect of aging on lees was different for the lactone family (only represented by γ-butyrolactone). The treatment resulted in a significant decrease in CLI 271 wines and no variations were observed in CLI 889 wines. In all cases, the concentrations of this compound were far from its perception threshold.

The increase of terpenes and norisoprenoids on lees presence is likely related to the release during autolysis of β-glucosidases. These enzymes are able to break the glycoside bound of these volatile compounds releasing the free aromatic form, which has an influence on final aroma of wine [33]. In wines aged on lees, varietals tended to increase in agreement with other works [26, 33]. Individually (Table 1), the varietal compounds β-myrcene, limonene and γ-terpinene varied significantly but were found in low concentrations. As for β-damascenone, a significant increase was only seen in CLI 889 wines with lees treatment always exceeding their olfactory perception threshold.

Regarding to acids content, the decrease in their concentration as a consequence of contact with lees had no effect on the aroma of the wines. The maximum values correspond to octanoic acid, and after that, to hexanoic acid in wines aging on lees in good agreement with other studies in white wines [26, 34].

According to the family of alcohols (Figure 2) only the wines made with strain CLI 271 aged on lees showed a notable decrease, 5 of the 6 compounds identified (Table 1) decreased significantly. Methionol and β-phenylethanol seem to actively contribute to the aroma of these wines with a concentration above their perception threshold even while decreasing in presence of lees. Isobutanol decreased significantly and was found at levels below its threshold in CLI 889 wines aged on lees. These changes in alcohol content compounds were similar to obtained in Macabeo wines [26].

3.2 Odor activity values of Malvasia aromatica wines

In order to estimate the sensory contribution of aromatic compounds to overall aroma of wine, the odor activity value (OAV) was calculated for all aroma compounds in study (Table 2). An aromatic compound contributes actively in wine when its concentration is higher than its olfactory perception threshold (OTH). The concentration/threshold ratio, known as odor activity value (OAV), shows the contribution of a specific compound to wine aroma properties. Those compounds with an OAV ≥ 1 can be considered active odorants [21].

				OAV^a
	Sensory descriptor	OTH^*	CLI 271	CLI 271 L	CLI 889	CLI 889 L
Linalool	Floral, citric	25	4.66	5.01	5.24	5.61
β-damascenone	Floral, lilac	0.05	24.80	23.27	22.13	26.60
Isoamyl alcohol	Vegetal/herbaceous	30	9.87	8.74	10.52	10.31
Isobutanol	Alcohol	40	<1	<1	1.06	0.94
Methionol	Cooked vegetable	1	1.80	1.36	<1	<1
β-Phenylethanol	Roses	14	4.02	3.21	2.45	2.22
Ethyl butyrate	Acid fruit, apple	0.02	13.68	9.97	15.33	13.76
Ethyl isovalerate	Sweet fruit, orange, blackberry	0.003	87.17	57.09	87.52	74.92
Isoamyl acetate	Banana	0.03	56.14	46.65	37.07	32.48
Ethyl hexanoate	Fruit, green apple	0.01	53.80	38.11	51.90	35.60
2-Phenylethyl acetate	Green apple	0.25	1.04	1.36	<1	<1
Butyric acid	Cheese	0.17	3.79	2.38	4.30	3.79
Isovaleric acid	Blue cheese	0.03	81.04	59.82	69.70	55.59
Hexanoic acid	Cheese	0.42	6.95	7.22	5.51	5.80
Octanoic acid	Butter, sour	0.50	8.01	8.43	6.55	6.46
Diacetyl	Butter	0.10	1.62	1.14	2.31	1.14

Table 2.

Odor threshold values and odor activity values of the volatile compounds with the greatest influence on the aroma of Malvasia wines fermented with S. cerevisiae CLI 271 and CLI 889 with lees treatment (L).

*OTH: Odor threshold values

^aOAV: Odor activity values calculated by dividing concentration by odor threshold value of the compound.

OTH and OAV are given in mg l⁻¹ except linalool and β-damascenone which are in μg l⁻¹. Sensory descriptor and OTH values according to the references included in Table 1.

In bold the compounds that seem to have a clear connection with aging on lees treatment.

Sixteen of thirty-five identified compounds presented OAV > 1, contributing to the aroma of the wines studied (Table 2). The varietal compounds linalool and β-damascenone had an active contribution to the aroma of all resulting wines providing floral aromas with citrus and lilac attributes respectively. The OAV was always higher than 1. β-damascenone also presented concentrations higher than OTH in all wines showing interesting correlations between the increase in OAVs of wines fermented with CLI 889 yeast strain aged on lees. A study realized by Gallardo-Chacón et al. [35] in Cava aged in contact with lees, they detected some norisoprenoids as β-damascenone more retained in lees surface and thus its presence in the volatile profile of Cava was significant.

The alcohols, characterized by vegetal and bitter aromatic descriptors with the exception of β-phenylethanol (roses), did not show significant differences according to aroma contribution. OAVs were higher than 1 in all wines except isobutanol in CLI 889 L wines.

The wine elaborated with S. cerevisiae CLI 271 strain obtained higher concentrations of 2-phenylethyl acetate above its OTH and presented OAV > 1 in traditionally preserved wines (non lees) increasing significantly after lees treatment. This compound is related to fruity attributes. Previous studies have demonstrated the capacity of this locally selected yeast strain CLI 271 to influence in the volatile profile of wines, increasing the concentration of fresh-fruity aroma compounds [15]. In general, esters decreased in both wines treated with lees but their OAVs remained above unity and actively contributed to aroma.

A significant decrease is observed in CLI 889 wines regarding diacetyl during aging on lees (Table 1). This compound is related to buttery aroma descriptors although their OAV is still higher than 1 (Table 2).

Finally, butyric, isovaleric, hexanoic and octanoic acids, characterized by their unpleasant odors (cheese and rancid attributes) influenced all the wines equally, all wines had OAV > 1. They cannot be considered determinants of the differences in the overall aromatic quality of the wines.

3.3 Sensory characteristics of Malvasia aromatica wines

A descriptive tasting of wines was performed at visual, olfactory and gustative levels in order to determine the main organoleptic differences among wines after aging on lees (Figure 3A–C). The sensory characterization of the Malvasia aromatica wines was obtained from the attributes defined by the tasters. The results were statistically treated and differences between wine samples with a significance level of 0.1% (p < 0.001) were considered very significant, of 1% (p < 0.01) considered significant and of 5% (p < 0.05) considered low significant.

Figure 3.

Descriptive analysis of Malvasia wines elaborated with S. cerevisiae CLI 271 and CLI 889 with lees treatment (L). A) Visual phase; B) Olfactory phase; C) Gustative phase.

In the visual phase (Figure 3A), aging on lees (L) resulted in Malvasia aromatica wines with significantly higher scores in vivacity/brightness descriptor. This could be related to the improved stability of white wines due to the action of some mannoproteins released by yeasts that help to reduce the turbidity produced by the presence of unstable proteins after fermentation [36, 37]. On the other hand, CLI 271 control wines were scored with higher color intensity with statistical significance in difference that wine treated with lees (CLI 271L). The influence of lees in wine color has been pointed out by several authors in artificial wines [38, 39], in white wines [40, 41] and red wines [42]. This fact may be due to capacity of yeast compounds such as mannoproteins and glucans, to interact with different wine phenolic molecules as total polyphenols, flavonols and tannins [43]. In accordance with [27] the use of lees can give rise to a reduction in the color intensity of wines after treatment, so they can be used as agents for reducing browning in white wines.

In the olfactory phase (Figure 3B), CLI 889L wines obtained significantly higher scores on floral aroma attribute. As shown in Table 1, wines elaborated with CLI 889 strain presented higher concentrations of varietals compounds and lees treatment could have contributed to enhance the floral characteristics of this family of volatiles. According to the results in Table 2, there was a noticeable increment of linalool and β-damascenone compounds. Regarding to CLI 271L wines were considered significantly fruitier than CLI 271 control (non lees). These results are in line with those obtained by Del Barrio-Galán et al. [27] in the sensory analysis of the Verdejo variety where the treatment with lees would have displayed stronger fruity and floral aromas and higher olfactory intensity than the control wines which could indicate that these initially retained aromatic compounds are released over time, increasing aroma intensities. Based on the analysis of Table 2, 2-phenylethyl acetate with fruit descriptor increased significantly after lees aging. Aging on lees decreased the vegetal character of Malvasia aromatica wines from both winemaking but without significant differences. The same behavior for this attribute was reported by other authors in white wines aged on lees [34]. Some tasters noted the presence of microbiological aromas in CLI 889 wines without lees, but this character was not detected in wines aged on lees. There were no significant differences between treatments in the rest of the parameters.

Finally, in the gustatory phase (Figure 3C), there are almost no differences between treated wines and their controls. Wines not aged on lees were evaluated as alcoholic and scored higher in global taste quality without significant differences. Aging on lees apparently reduced bitterness in Malvasia wines, but the decrease was only significant in CLI 271. Additionally, there was a tendency for the lees-treated wines to have greater structure and less acidity, although this was not statistically significant. This trend toward a bitterness and acidity reduction and an increment of mouthfeel and balance in wines treated with lees has been described in previous publications [27, 44].

With the aim of completing the sensory study, triangular tests were done to determine whether descriptive analysis was determinant to differentiating the samples. Discriminant triangular tastings were carried out using dark glasses and two series of tastings were performed by each type of wine: CLI 889 vs. CLI 889 L, CLI 271 vs. CLI 271 L. The panel clearly differentiated CLI 271 and CLI 889 wines aged on lees from their respective controls with statistical significance levels of 1% and 0.1% in the two tests performed for CLI 271 wines, and 5% and 1% in the two tests performed for CLI 889 wines. In addition, tasters were asked to assess their hedonic preference for wines in the tests presented, taking into account the preferences of the taster who answered correctly. The tasting panel showed preference by CLI 271 wines in 57% of cases in the first test and 62% in the second. Preferences for the CLI 889 wines were equally divided. In the first test, 50% of the correct judges chose CLI 889 L samples. In the second test, 57% of the correct tasters preferred CLI 889 L.

4. Conclusions

According to the results, we can affirm that lees treatment has had an effect on the result of aroma and sensory composition of Malvasia aromatica wines. Some volatiles showed significant variations after lees treatment. Esters were the most affected compounds along with carbonyl compounds in wines fermented with CLI 889, decreasing in concentration after aging on lees. Wines from strain CLI 271 suffered the greatest losses in the alcohol family. According to the yeast strains used for winemaking, we can conclude that aging on lees has caused greater changes in the volatile composition of CLI 889 strain wines. The results of the sensory analysis indicate that Malvasia wines aged on lees have more vivacity and brightness than those aged conventionally, also could reduce acidity, bitter and vegetal character of the wines. The treatment with lees enhanced the floral character of the Malvasia aromatica wines fermented with the native yeast strain CLI 889 and the fruity character of the CLI 271. Aging on lees seems to have a positive effect on wines from the native yeast strains CLI 889, since they were preferred over those conventionally aged. In the case of CLI 271 wines, the similar development of wine with and without lees suggests that factors determining aroma and flavor development are influenced by S. cerevisiae CLI 271 fermentations. Probably, it could be interesting to test other aging on lees times in CLI 271 wines to find out more differences after lees treatment. Therefore, the employment of aging on lees in wines fermented with locally-selected S. cerevisiae can be used to obtain quality wines from the alternative variety Malvasia aromatica on PDO “Vinos de Madrid”.

Acknowledgments

JMC acknowledges INIA for a PhD grant (FPI-INIA 2012 call). The authors acknowledge IMIDRA colleagues at the El Socorro experimental farm and the Microbiology lab for providing material of Malvasia aromatica and yeast strains respectively used in this work and IMIDRA for its financial support.

Conflict of interest

The authors declare no conflict of interest.

Acronyms and abbreviations

PDO	Protected Designation of Origen
L	lees
DVB	divinylbenzene
CAR	carboxen
PDMS	polydimethylsiloxane
GC-FID	gas chromatography with flame-ionization detection
GC/MS	gas chromatography–mass spectrometry
HS-SPME	headspace-solid phase microextraction
C13	C13-norisoprenoids
ANOVA	analysis of variance
OTH	olfactory perception threshold
OAV	odor activity value