Aromatic Compounds in Wines Produced During Fermentation: Effect of Three Red Cultivars

Abstract

The aromatic compounds produced during the fermentation of the red grape cultivars Caiño Tinto, Caiño Longo, and Caiño Bravo were analysed by gas chromatography (FID) on the wines of 2002 and 2003 vintages. In both years, significant differences (p < 0.001) were observed between the wines with respect to the concentrations of aromatic compounds. Caiño Longo wines had the highest concentrations of acetates and esters. The concentrations of ethyl ester and acetates in Caíño Bravo wines were comparatively very low. Principal components analysis confirmed these results: the wines made from the different cultivars and the vintages were clearly different.

Keywords:

• Caíño Longo
• Caíño Tinto
• Caíño Bravo
• wine aroma
• Gas Chromatography
• Principal Components Analysis

INTRODUCTION

Galicia, in the northwes of Spain, has a long wine-making tradition one. Proof of this is that almost all the region's vineyards lie within one of its five Appellation Contrôlée areas (Ribeiro, Rías Baixas, Monterrey, Valdeorras, and Ribeira Sacra). In recent years there has been a trend towards recovering the use of native Galician grape cultivars, whose presence had become reduced. These cultivars are well adapted to the area and transmit to their wines the characteristics of the climate and soil in which they are grown. Although currently it is produced only in small quantities, the red cultivar Caíño Tinto is one of the most appreciated of the Rías Baixas and Ribeiro Appellation Contrôlée areas, the two most important in Galicia. Caíño Bravo and Caíño Longo are also traditional red cultivars of the region.[1] The names of Caiño Longo and Caiño Bravo appear in the List of Commercial Vine Varieties and Rootstocks (Lista de Variedades Comerciales y Portainjertos de Vid[2] as “provisional inscriptions.” They will become definitive inscription when thre was more information available on their ampelographic and oenological characteristics. For a cultivar to be officially cultivated in any Spanish winemaking region, or for it to be introduced into an Appellation Contrôlée area as an authorised or preferred variety, its name must appear as a definitive inscription in the above list.

It is of great importance in studies designed to differentiate grape cultivars, to determine the volatile composition of wines. According to Rapp,[3] the presence of several hundred volatile compounds belonging to different chemical families, however, influenced the aroma of wine. The higher alcohols are the most common, followed by ethyl esters of fatty acids.[4] Nevertheless, the initial composition of the must,[5] the yeast strain used,[6] and the winemaking process[7,8] could cause quantitative changes resulting in different sensorial profiles by which wines can be classified.[9,10] Higher alcohols and esters are originated during the alcoholic fermentation and their final concentration depend on the type of grape and yeast strain used.[11,12] Synergy exists between the grape cultivar and yeast metabolism – something that should be borne in mind when is not planed inoculation of commercial yeast. Rapp[3] indicates that while the higher alcohol and ester contents could be used to evaluate winemaking technologies, they are insufficient for varietal characterization purposes. Aleixandre et al.[13] attempted to differentiate wines made from different grape cultivars in terms of chemical factors such as colour, total and volatile acidity, alcohol content, and reducing sugar content etc.

Esters originated during fermentation contribute positively to the overall aroma of wines, especially those made from less aromatic grapes. Short chain esters are the most important esters since they provide a fruity aroma.[14] Higher alcohols can also supply fruity notes, but they should not surpass 300 mg/L;[15] higher concentrations are considered to be negative influence the final aroma. In red wines, maceration time has to be controlled since, according to Palacios et al.,[16] it is during this process when the higher alcohol content increases. Temperature should also be controlled; fermentation at room temperature leads to lower concentrations of higher alcohols than when this process proceeds at cooler temperatures.[17] The objective of the present work was to identify the aromatic compounds produced during the microfermentaiton of musts from Caíño Tinto, Caíño Bravo, and Caíño Longo grapes.

MATERIALS AND METHODS

Musts

The grapevine cultivars used in this work belong to the collection of the Misión Biológica de Galicia — CSIC, maintained in field in Salcedo (Pontevedra, Spain). The mean anual temperature is 14.4°C, and the mean annual rainfall 1586 mm (with strong annual fluctuations). All the vines (10 replicates per cultivar) were of the same age (10 years). All vines were grafted onto 110-Ritcher rootstocks, grown en espalier, subjected to Sylvoz pruning and all received the same crop protection treatments. During the growth season weeds were removed manually when necesary.

Fermentation

In 2002 and 2003, Caíño Bravo, Caíño Tinto, and Caíño Longo grapes were harvested and crushed by hand (to prevent the breakage of the seeds). SO₂ was added at 50 mg/L. Spontaneous fermentations (at 18°C for 15 days) were performed in 16 L glass vessels, individually containing 10 L of the must of each cultivar. The specific density of the developing wine was measured daily by hydrometry. When fermentation completes the wine was separated from the skins. In 2003, it was possible for the wines to undergo malolactic fermentation after finishing alcoholic fermentation. All wines were racked and the free SO₂ content adjusted to 50 mg/L. After bottling, 1L of each wine was conserved at 10°C for an analysis 30 days later.

Chemical Analysis

The composition of wines was determined according to official methods.[18] The pH was measured using a pH meter; the ethanol content was measured by distillation of the wine (made alkaline by a suspension of calcium hydroxide); total acidity was measured by titration (using bromothymol blue as an indicator); volatile acidity was measured by titration of the volatile acids separated from the wine by steam distillation; total dry extract was measured by measurement with a densitometer; tartaric acid content was measured by the gravimetric method; malic acid content was measured by an enzymatic method; reducing sugar content was measured by enzymatic determination of the glucose and fructose concentrations; and the total polyphenol content was measured using the Folin Ciocalteu index.

Volatile Compounds

The contents of the most important aromatic compounds produced during fermentation (i.e., higher alcohols, esters, and acetates) were determined by gas chromatography (GC), using a Hewlett Packard 5890 Series II gas-chromatograph equipped with a flame ionisation detector. The compounds were separated in a Chrompack CP-Wax 57CB fused-silica capillary column (50 mm × 0.25 mm i.d; 0.25 μm film thickness; polyethylene glycol stationary phase). The analytical conditions were: injector temperature 250°C, detector temperature 260°C, carrier gas was helium at 1.07 mL/min, and make-up gas was nitrogen 30 mL/min. The detector gas flow rates were: hydrogen, 40 mL/min, air, 400 mL/min.

Due to their high concentrations in wines, methanol and the higher alcohols contents were determined by adding 1 mL of an internal standard solution (1 g of 4-methyl-2-pentanol per 1 L of ethanol) to 10 mL of each wine sample prior to analysis. A 2 μL aliquot of this sample was injected directly and split 1:1. The operating temperature was set at 60°C for 15 min before being raised to 200°C at a rate of 3°C/min.

The extraction of the esters and acetates was performed according to the method of Bertrand:[19] 2 mL of 3-octanol (50 mg/L) were used as an internal standard; 1 mL of sulphuric acid (1/3) was added to 50 mL of wine. Each sample was subjected to extraction three times with 4, 2 and 2 mL of diethyl ether-hexane (1:1, v/v). 1 μL of the organic extract was, then, injected into the chromatograph in splitless mode (30 s). The operating temperature was set at 55°C for 15 min and then it was raised to 200°C at a rate of 3°C/min. The wine aroma components were identified by comparing their retention times with those of pure standards. An internal standard was used to allow quantification.

Statistical Analyses

Differences among wines assessed by one-way ANOVA. Principal component analysis (PCA) was, then, performed using SAS software.[20]

RESULTS AND DISCUSSION

All the fermentations showed typical fermentation curves (Fig. 1). All three musts of the 2002 vintage showed a 5-day delay in starting fermentation. Table 1 shows the physico-chemical characteristics of the different wines. The alcohol content of all three was low, indicating that the commercial scale used for these grape cultivars might require their mixing with other types of grape. The three wines had a high malic acid content, especially Caíño Bravo; Therefore, malolactic fermentation might have improved the taste of the final wine.

Figure 1 Fermentation curves for Caiño Tinto, Caiño Longo, and Caiño Bravo wines. A) Vintage 2002 and B) Vintage 2003.

Table 1 Values for classic wine variables

	Caíño Longo		Caíño Tinto		Caíño Bravo
	2002	2003	2002	2003	2002	2003
Ethanol (% v/v)	9.46	9.70	9.16	9.30	7.86	8.20
Total acidity (g tartaric acid /L)	9.30	8.50	9.10	5.90	10.30	7.00
Volatile acidity (g acetic acid/L)	0.30	0.20	0.30	0.50	0.30	0.40
Reducing sugars (g /L)	0.70	1.30	0.70	1.30	1.00	0.60
Tartaric acid (g/L)	2.20	1.70	2.40	1.30	2.60	1.30
Malic acid (g/L)	4.00	0.50	5.30	0.33	6.80	0.30
Total polyphenols	35.81	27.71	36.19	22.85	55.66	26.38

The reducing sugar contents of wines of both vintages showed them to be dry. The low volatile acid contents of the 2002 wines showed that the fermentation process had proceeded correctly; the higher content of the 2003 wines was due to the fact that they underwent malolactic fermentation after completing alcoholic fermentation. From an oenological point of view, the Caíño Tinto wines had the most potential. After malolactic fermentation wines were balanced. However, the Caíño Bravo and Longo wines showed very high acidity levels even after this process. This, along with their low alcohol content, renders them rather destructured.

Table 2 shows the results of the analysis of the concentrations of aromatic compounds. The methanol content of all three wines was significantly different in both vintages, and much higher than the values reported in the literature for wines made from other grape cultivars.[16] Methanol is not a fermentation product but it derives from the action of pectolytic enzymes, therefore, its concentration in wine does not depend upon the fermentation conditions, but rather upon the maceration process and the amount of endogenous pectolytic enzymes in the grapes. Since the must was in contact with the skins until the end of alcoholic fermentation the necessary enzymatic activity was high. Malolactic fermentation had no influence on the methanol content; the three wines of the 2003 vintage showed different methanol concentrations (Table 2).

Table 2 Mean concentrations (mg/L) and standard deviations of the volatile compounds in Caiño Longo, Caiño Tinto, and Caiño Bravo wines,in 2002 and 2003 vintages

	2002				2003
Compound	Caiño Longo	Caiño Tinto	Caiño Bravo	Sig.	Caiño Longo	Caiño Tinto	Caiño Bravo	Sig.
Alcohols
1-Propanol	6.74 ± 0.38	7.94 ± 0.33	10.75 ± 0.87	∗∗∗	36.89 ± 0.88	30.45 ± 0.55	35.03 ± 1.61	∗∗∗
2-Methyl-propanol	131.98 ± 0.89	125.81 ± 1.21	154.35 ± 5.03	∗∗∗	43.18 ± 2.39	80.63 ± 1.86	38.19 ± 0.39	∗∗∗
2-Methyl-butanol	54.53 ± 0.61	53.08 ± 0.91	57.17 ± 2.19	∗	30.57 ± 1.01	47.21 ± 0.37	18.39 ± 0.43	∗∗∗
3-Methyl-butanol	187.00 ± 2.52	177.92 ± 1.04	200.92 ± 5.65	∗∗∗	132.22 ± 1.38	166.77 ± 3.05	64.21 ± 1.02	∗∗∗
Methanol	88.69 ± 3.40	135.62 ± 1.76	214.87 ± 3.38	∗∗∗	168.83 ± 2.68	268.84 ± 9.58	165.43 ± 2.37	∗∗∗
Esters and Acetates
Ethyl butyrate	0.44 ± 0.03	0.31 ± 0.06	0.38 ± 0.00	∗	0.12 ± 0.01	0.70 ± 0.01	0.14 ± 0.00	∗∗∗
Ethyl hexanoate	0.22 ± 0.02	0.24 ± 0.02	0.18 ± 0.00	∗	0.27 ± 0.00	0.20 ± 0.02	0.13 ± 0.01	∗∗∗
Ethyl octanoate	0.23 ± 0.01	0.21 ± 0.02	0.13 ± 0.00	∗∗∗	0.47 ± 0.04	0.42 ± 0.03	0.25 ± 0.01	∗∗∗
Ethyl decanoate	0.03 ± 0.00	0.03 ± 0.00	0.04 ± 0.00	ns	0.08 ± 0.00	0.11 ± 0.00	0.05 ± 0.00	∗∗∗
Diethyl succinate	2.71 ± 0.26	2.55 ± 0.01	1.53 ± 0.04	∗∗∗	0.66 ± 0.04	0.52 ± 0.00	0.55 ± 0.00	∗∗∗
Ethyl lactate	6.22 ± 0.48	7.46 ± 0.32	8.76 ± 0.34	∗∗∗	31.44 ± 0.54	66.40 ± 3.84	22.86 ± 0.90	∗∗∗
Ethyl myristate	0.16 ± 0.00	0.15 ± 0.01	0.11 ± 0.01	∗∗	n.d.	n.d.	0.03 ± 0.00	∗∗∗
Isoamyl acetate	0.12 ± 0.00	0.11 ± 0.02	0.09 ± 0.00	∗	0.27 ± 0.01	0.20 ± 0.01	0.13 ± 0.00	∗∗
Hexyl acetate	0.21 ± 0.01	0.24 ± 0.02	0.17 ± 0.01	∗	0.55 ± 0.00	0.42 ± 0.00	0.25 ± 0.00	∗∗∗
Ethyl acetate	28.51 ± 0.78	22.88 ± 1.41	58.90 ± 3.37	∗∗∗	69.21 ± 1.78	125.57 ± 3.44	58.01 ± 2.93	∗∗∗
Data are means of triplicates. n.d. = not detected. Sig: ∗, ∗∗, ∗∗∗ = significance at p < 0.05, p < 0.01, and p < 0.001, respectively.

Higher alcohols and esters, produced during alcoholic fermentation, play an important role in the flavour of wines, depending on the types of compounds and their concentration.[21] The higher alcohol contents of the three wines were significantly different (p < 0.001) in both vintages; however, 3-methyl-butanol was the most common. In 2002, Caíño Bravo produced the wine with the highest concentration of higher alcohols, while Caíño Tinto produced the lowest concentrations. However, in all wines the final concentration of higher alcohols was above the 300 mg/L level set by Rapp and Versini.[15]

In the 2003 wines, after malolactic fermentation there was a generally low concentration of higher alcohols. This agrees with the results of Agouridis et al.[22] However, unlike that reported by the latter authors, the 1-propanol content increased. In 2003, Caíño Tinto produced the highest concentration of higher alcohols, while Caíño Bravo produced the lowest. Following malolactic fermentation the level was always below 300 mg/L (except for Caíño Tinto); this would improve the sensorial quality of wines.

The esters are the most important compounds in young wine aroma and thet are among key compounds in the fruity flavours of wines.[23] With the exception of ethyl decanoate in the 2002 vintage, the ester and acetate contents of the wines were significantly different (Table 2). The majority of esters were found in great concentration in the Caíño Longo wines, making them fruity. Ethyl acetate was the most common ester but significantly different concentrations were recorded for the three wines in both years and malolactic fermentation increased its concentration, especially in Caíño Tinto wine, which also showed a high ethyl lactate content. This increase after malolactic fermentation has also been reported by Agouridis et al.[22] For the remaining esters, the most important changes after malolactic fermentation were the increase in the content of medium chain-length ethyl esters (responsible for fruity notes) and the fall in diethyl succinate and myristate concentration, that notably improve the organoleptic qualities of the wine. The Caíño Longo wine should be the most aromatic wine since it contained significantly more acetates and esters than the others. The Caíño Bravo wine had the lowest concentration of ethyl esters and acetates, therefore, it should therefore be the least aromatic.

PCA was used to identify the aromas that discriminated best among the wines of the different varieties. The first two principal components accounted for 82% of the total variance (60.5 and 21.5%, respectively). The first principal component (Prin1) was characterised by 1-propanol, ethyl butyrate and ethyl octanoate attributes with a positive loading and 2-methyl-1-propanol, ethyl succinate and ethyl myristate with negative loading. For the second principal component (Prin2), the attributes 2-methyl-1-butanol, 3-methyl-1-butanol, methanol, isoamyl acetate and ethyl acetate all showed positive loading. Figure 2 shows the good separation between the 2002 and 2003 vintages. In conclusion, significant differences were seen between the three wines in terms of the concentration of most aromatic compounds; this was the case in both vintages. The Caiño Longo wines had the highest concentration of acetates and esters and therefore, they are the most aromatic.

Figure 2 PCA of aromatic compounds in Caiño Tinto, Caiño Longo, and Caiño Bravo wines.

CONCLUSION

Wine aroma components were identified in red wines (vintages 2002 and 2003) made from Caíño Tinto, Caíño Bravo, and Caíño Longo. From an oenological point of view, the Caíño Tinto was the most interesting wine because its composition was the most equilibrated. Significant differences were found for all aromatic compounds except ethyl decanoate in 2002 vintage. The Caíño Longo wine should be the most aromatic wine since it contained significantly more acetates and esters than the others. The PCA showed a good separation of the wines and the different vintages. The vintage of 2002 was more homogeneous between wines than 2003 vintage.

ACKNOWLEDGMENTS

This work was supported by the Spanish Ministry of Science and Technology (VIN00-036-C6-3, HP2000-0032, RF02-004-C5–2), Diputación Provincial of Pontevedra and Xunta de Galicia (Spain). M. Vilanova was supported by a I3P contract financed by the CSIC- European Social Fond.

Notes

2. Boletin Oficial del Estado. Orden APA/748/2002, del 21 de marzo, por la que se dispone la inscripción de variedades y portainjertos de vid en la Lista de Variedades Comerciales de Plantas, 2002, 84, 13,351–13,353.

18. European Union Commission Regulation (EEC) No. 2676/90 of 17 September 1990, determining Community methods for the analysis of wines.