Organic acids composition of Patagonian Pinot noir grapes and wines obtained from guided (GF) and spontaneous (NF) winemaking (extracted from Curilén et al. [22]).
Abstract
In young wines, the compounds responsible for wine flavor come from two possible origins: grapes and microorganisms involved in winemaking. Yeasts play the most important role in flavor influence because of their role in conducting the alcoholic fermentation (FA), the key process of winemaking. Ecological studies show that yeast diversity is significantly influenced by geographical and technological features of each particular winegrowing region. Wines from Argentine have achieved high-quality certifications, and particularly, in the Comahue region, wine production is mainly oriented to young red wines varieties, some of which found in this region optimal ecological condition to express all their enological potential. Despite this, the need to satisfy the demands of an increasingly competitive and globalized international market and the consumer demand for new wine styles with the best quality/price ratio imposes the regional productive sector new challenges that require technological innovation. The use of starter cultures developed from indigenous yeast isolated from our region, specially selected for its enological properties, appears as a valuable tool for differentiation, diversification, and quality improvement of wines. In this context, conventional and non-conventional yeasts were isolated and selected over the years and used for vinifications in red grape varieties (Pinot noir and Malbec). Assays were carried out at laboratory and pilot scale, in the 2010–2015 vintages. The experiences developed along the years contribute to a better understanding of the processes involved in the production of improved wines by autochthonous strains, an important practice to develop a more competitive regional wine industry.
Keywords
- wine flavor
- fermentation
- Saccharomyces cerevisiae
- non-Saccharomyces yeasts
- mixed starters
- microbial interactions
- indigenous biota
1. Introduction
Wine chemical composition is the foundation for its sensorial features, as color, appearance, body, flavor (aroma and taste), as well as its mouth and palate sensations [1]. Among these, wine flavor is a key attribute for quality and choice from consumers. The flavor of wine is a sensory perception that varies with the individual, the context of the consumer experience, and the chemical composition of the product [2, 3]. The wine chemical composition is determined by many factors such as grape variety, the geographical and viticultural conditions of grape cultivation, the microbial ecology of the grape and fermentation processes, and winemaking practices [4]. In young wines (without aging), like the ones mainly produced in the Patagonia Argentina, the compounds responsible for wine flavor come from two possible origins: grapes and microorganisms involved in winemaking, mostly yeast and to a lesser extent lactic acid bacteria (LAB) [5]. Grapes contribute with varietal aroma compounds, such as floral monoterpenes or volatile thiols, among others [1]. Among the microorganisms, yeasts play the most important role in flavor influence because of their role in conducting the alcoholic fermentation (AF), the key process of winemaking. During AF yeasts transform grape sugars and other components to ethanol, carbon dioxide, and different primary metabolites that confer a particular character to wine, but they also contribute with minority volatile compounds involved in determining the fermentative or secondary aroma [1, 2, 6, 7]. These compounds (esters, higher alcohols, carbonyls, short-chain fatty acids and sulfur compounds) arise from the metabolism of sugars and amino acids from the must, and their quality and content depends on the ecology wine yeast associated with the process [1]. Finally, when needed, LAB through malolactic fermentation not only provides wine deacidification, but can also enhance its flavor profile [1, 2, 8].
In Argentina, wine production has historically occupied a place of importance in the agricultural industries. The country is currently the fifth largest producer of wines, the seventh consumer, and the tenth largest exporter. There are two ways of imposing a wine in the market, highlighting its grape quality, that is,
North Patagonian wine production is mainly oriented to elaboration of young dry wines from red grape varieties (80%) some of each, as Pinot noir and Merlot, have found in this region the optimal ecological conditions to express all their enological potential [11]. Additionally, important volumes of Malbec grapes, the
Actually, regional wine production is based on both spontaneous alcoholic fermentations of the grape musts or conducted fermentations using commercial yeast starters. However, commercial starters for alcoholic fermentation found actually in the market are composed by yeast strains isolated from the most important winegrowing areas in the world, except Argentina. Given the significant influence that the biota of yeast has on the aromatic quality of young wines elaborated from aromatically neutral grapes, wine style produced mostly in Patagonia, development of starter cultures consisting of yeast strains isolated from the own region (indigenous yeasts) appears as a valuable tool to improve the quality of Patagonian vitiviniculture, upgrading its capacity of commercial competition in domestic and international market.
1.1. The microbial ecology of Patagonian red winemaking
Winemaking is a complex microbial ecosystem that involves interactions between filamentous fungi, yeasts, and bacteria with different physiological and metabolic characteristics [3, 13, 14]. Ecological studies have shown that this microbial diversity is significantly influenced by geographical and technological features of each particular winegrowing region, which defines the terroir. To understand how this microbial terroir contributes to the natural environment of vineyard and how it imprints differential character of wine required to know all processes associated with winemaking which start at the harvest of grapes and then evolve throughout fermentation process [15–17].
Microbiological studies carried out during several years in the Patagonian region allowed to characterize the biota associated with grapes [18–22], cellars [23, 24], and red vinification environments [19–22, 25, 26], (Figure 2). Yeasts associated with spontaneous wine fermentations come from two possible origins: grapes and cellar surfaces. Several factors such as development stage and sanitary state of the berries, the climate (particularly temperature and rainfall), water availability, direct exposure to sunlight, use of agrichemicals, grape vine canopy management system as well as nature, cleaning and sanitization of equipment surfaces, nature, cleaning and sanitization of equipment surfaces, among others, as well as certain enological practices could affect the yeast community composition on grapes and cellar surfaces affecting the kinetics of yeasts growth during fermentation [3, 14, 27]. In this context, in all cases, ripe, whole and healthy grapes from Merlot, Malbec, and Pinot varieties were gathered by random sampling (n = 536) from vineyards associated with cellars which is noted in Figure 1 for 1993–1998 and 2005–2009 vintages at harvest time, and cellar sampling was carried out on the internal walls of the fermentation vats from the same cellars approximately four weeks before harvest. Yeast samples from grape surfaces were obtained by agitation followed sonication of each grape in pure and sterile water. Additionally, samples (1L) of Malbec, Merlot, and Pinot noir fermentation musts samples were taken in duplicates during spontaneous alcoholic fermentation at the initial (14°Bé), middle (6°Bé), and end (≌2 g/L TRS) stages in the same cellars described above. All yeasts were isolated on GPY agar (composition in g/l: yeast extract 10, glucose 20, peptone 20, and agar 20; pH 4.5 supplemented with 100 ppm of ampicillin) plates, and they were identified according to the methods and keys proposed by Kurtzman and Fell [28] and by PCR-RFLP analysis of the ITS1-5,8S-ITS2 region from the nuclear rDNA gene complex [29]. Results of these studies evidence that yeast biota associated with grape surfaces are mostly aerobic (57%), while the one associated with cellar surface is mostly facultative (64%). These results of yeast diversity obtained from initial musts are consistent with the hypothesis proposed on origin of yeasts musts (Figure 2). On the other hand, this study also evidences that
With this greater knowledge, alcoholic fermentation is now seen as a key process where winemakers can creatively engineer wine character and value through better yeast management and can strategically tailor wines to a changing market [17].
Malolactic fermentation (MLF) is other opportunity to modulate the aroma of wine [1, 42]. MLF, the decarboxylation of L(−)malic acid to L(+)lactic acid, is an important secondary fermentation carried out by lactic acid bacteria (LAB) during the vinification of most red wine styles as those elaborated in Patagonia. Malic acid, together with tartaric acid, is the most important constituents of organic nonvolatile acid fraction in grapes and grape musts, accounting for 90% of the titratable acidity and imbalances in this fraction can affect the physicochemical and sensory properties of wine, mainly mouthfeel [43–48]. In addition to deacidification, MLF can increase microbiological stability [13, 49] and enhance wine flavor and/or complexity [1, 50] but off-flavors as well as dangerous health compounds could also be formed [45]. Although
Several factors such as grapevine variety, vineyard agricultural practice, temperature, humidity and berry maturity degree, among others, may affect organic nonvolatile acid concentration in grape musts [44, 65, 66]. In particular, L(−)malic acid content, directly related to respiratory quotient of berries, is higher in grape musts from cooler regions than the ones from warmer regions [67]. In the Comahue region, one of the southernmost winegrowing regions of the world, malic acid concentrations account for the 56% of red grape must titratable acidity reaching the 66% in Pinot noir [19] the emblematic regional vine variety [11]. Additionally, to its contribution to wine acidity, malic acid represents a fermentable substrate for other microorganisms which can spoil the wine before and after bottling [68]. Without adjustment of acidity, the wines will be regarded as unbalanced or spoilt [1]. For these reasons, MLF is a routine enological practice in the Patagonian red winemaking and yeast-LAB interactions are a great concern for winemakers and researchers.
Organic acid * | Grapes | Wines | P value* | |||
---|---|---|---|---|---|---|
GF | NF | |||||
Running wine | Bottled | Running wine | Bottled | |||
L(−)malic | 2.20 ± 0.66a | 2.08 ± 0.02ab | 2.01 ± 0.01b | 1.16 ± 0.02c | 0.97 ± 0.01d | <0.001 |
DL lactic | Nd | 0.84 ± 0.00a | 0.58 ± 0.00b | 0.97 ± 0.09c | 1.12 ± 0.02d | <0.001 |
L(+)lactic | Nd | 0.25 ± 0.03a | 0.30 ± 0.01a | 0.55 ± 0.04b | 0.75 ± 0.04c | <0.001 |
Citric | 0.78 ± 0.11a | 1.06 ± 0.04a | ndb | 0.83 ± 0.14a | ndb | <0.001 |
Succinic | Nd | 1.50 ± 0.12a | 0.96 ± 0.16b | 1.07 ± 0.13ab | 0.86 ± 0.05b | <0.001 |
In Patagonian winegrowing region, potential yeast-LAB interaction was studied in Pinot noir wine fermentations carried out at pilot [22] and industrial scales [8]. In all cases, malolactic fermentations were carried out spontaneously, whereas alcoholic fermentations were carried out in both, spontaneous (NF, carried out by indigenous yeast biota) and guided (GF, carried out by
2. Development of patagonian yeast starters
The practical consequence of studies on microbial ecology in winemaking was the development of starter cultures of AF. The use of commercial starter cultures for the FA in enology, with selected cultures of
The inoculation of grape musts with commercial starters was an enological practice strongly resisted by wine producers from Europe. The solidest argument for this resistance referred to the sensory quality standard, where flattened aromatic profiles were produced in each wine fermented with those starters. In modern wineries, the use of commercial starter culture to steer fermentations is being doubted, since they often lack of some advantageous enological traits, which are present when the spontaneous fermentation is ruled by indigenous populations [6]. For that reason, the exploitation of indigenous strains biodiversity has great importance for the characterization and selection of strains with peculiar phenotypes [17, 36]. While the preservation of spontaneous microflora is essential to obtain the typical flavor and aroma of wines deriving from different grape varieties [5, 13], the development of starter from
Microbiological studies carried out during several years in the Patagonian region described above allowed constituting an important collection of organisms relevant for enological application and wine starter elaboration. Our enology applied studies were divided into two basic objectives: isolation of indigenous
2.1. Saccharomyces cerevisiae indigenous starter
When indigenous F8 and commercial F15
In order to evaluate the capacity of the indigenous starter to dominate the fermentations, the dynamics of the
On the other hand, physicochemical analysis of the wines obtained during different years from Pinot noir, Merlot, and Malbec varieties were highly similar between both inoculated strains, where every product was considered acceptable for local young wines [26]. Nonetheless, sensorial analysis carried out by experts and consumers using qualitative and quantitative tests, respectively, showed significant differences between F8 and F15 wines.
Qualitative analysis was performed by a panel of experts using descriptive tests. As a whole, the global quality scores obtained in this analysis by F8 Pinot noir (68 = good) and Merlot (6.6 = pleasant) wines were higher than those obtained by F15 Pinot noir (52 = correct) and Merlot (5.7 = slightly pleasant) wines. Particular descriptions evidenced that Pinot noir F8 wines had good color intensities (showing a red color typical for the variety) and aromas of red fruits (cherries) with notes of sherry. In mouth, they were described as middling fruity, slightly rusty, sweet alcoholic. Meanwhile, Pinot noir F15 wines showed limpid and bright aspect and an intense reduced aroma that did not disappear with agitation. In mouth, they were perceived as slightly fruity and bitter, astringent, and tannic. On the other hand, both F8 and F15 Merlot wines showed a limpid and bright aspect and an intense brick red color, but the F8 aroma was more intense than the F15 aroma, being both aromas of medium quality. Pepper, red fruits, butter, leather, spice, and vanilla were the aromatic descriptors highlighted in the former and green pepper, cooked red fruits, spices, and pepper were described for the later. In the mouth, both wines showed good acidity and body, and they were persistent. However, F8 wines were described as round and equilibrated while F15 wines showed a tart taste [26].
At last, F8 Pinot noir and F8 Merlot wines were the favorite for the consumers (p < 0.05) with 72 favorable responses out of 119 questioned and 13 favorable responses out of 17 questioned, respectively, compared with their F15 controls according to the paired-preference test.
2.2. Non-Saccharomyces indigenous starter
One of the purposes of the study was to select autochthonous yeasts with metabolic ability to degrade L-malic acid for its potential use in equilibrated young wine elaboration. A total of 57 Patagonian non-
Figure 6 shows the results obtained from vinifications guided by pure cultures indigenous
Finally, sensorial analysis evidenced significantly differences in aromatic perception between
3. Conclusions
The extension of the selection of yeast for enological use among
Acknowledgments
This work was supported by grants from Universidad Nacional del Comahue (Programa de Investigación 04/L003 Desarrollo de Tecnologías y de Productos de Interés para la Industria Agroalimentaria) and MINCyT (PICT SU 2804/12 Levaduras y Bacterias Lácticas para la diferenciación de vinos Patagónicos).
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